Compositions and methods for treating retinal diseases

ABSTRACT

Disclosed herein are compositions and methods for treating, ameliorating or preventing a retinal disease or condition; improving a photopic (day light) vision; for improving correcting visual acuity, improving macular function, improving a visual field, or improving scotopic (night) vision by administration of retinal progenitor cells. The subject matter described herein also provides cell populations comprising retinal progenitor cells and methods of isolation thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This United States utility patent application is a divisional of U.S.utility patent application Ser. No. (USSN) 14/715,464, filed May 18,2015 (now pending), which is a divisional application of U.S. utilitypatent application Ser. No. (USSN) 14/118,223, filed Feb. 18, 2014 andissued as U.S. Pat. No. 9,107,897 on Aug. 18, 2015, which is a § 371national phase of PCT international patent application no.PCT/US2012/038342, having an international filing date of May 17, 2012,which claims benefit of priority to U.S. Provisional Patent ApplicationSer. No. 61/487,419, filed May 18, 2011. The aforementioned applicationsare expressly incorporated herein by reference in their entirety and forall purposes.

TECHNICAL FIELD

The subject matter described herein relates generally to the fields ofstem cell biology and regenerative medicine. In particular, the subjectmatter disclosed herein provides compositions and methods for treating,ameliorating or preventing a retinal disease or condition; improving aphotopic (day light) vision; improving or correcting visual acuity,improving macular function, improving a visual field, or improvingscotopic (night) vision by administration of retinal progenitor cells.The subject matter described herein also provides cell populationscomprising retinal progenitor cells and methods of isolation thereof. Inalternative embodiments, provided herein are compositions and methodsfor treating, ameliorating or preventing a retinal disease or condition,e.g., an Usher's disease, retinitis pigmentosa (RP), a degenerativeretinal disease, an age related macular degeneration (AMD), a wet AMD ora dry AMD, geographic atrophy, a retinal photoreceptor disease, adiabetic retinopathy, cystoid macular edema, uveitis, a retinaldetachment, a retinal injury, macular holes, macular telangiectasia, atraumatic or an iatrogenic retinal injury, a ganglion cell or opticnerve cell disease, a glaucoma or an optic neuropathy, an ischemicretinal disease such as retinopathy of prematurity, retinal vascularocclusion, or ischemic optic neuropathy; or improving a photopic (daylight) vision; or for improving correcting visual acuity, or improvingmacular function, or improving a visual field, or improving scotopic(night) vision. In alternative embodiments, provided herein areheterogeneous mixtures of fetal neural retinal cells and methods andcompositions (e.g., kits, formulations and the like) for making andusing them.

BACKGROUND

Retinal degenerations are a heterogeneous group of eye diseases thatresult in the permanent loss of vision and affect millions ofindividuals worldwide. Although the molecular mechanisms underlyingthese conditions vary, they share a common endpoint: the irreversibledeath of the photoreceptor cells. No effective treatment is currentlyavailable to restore lost photoreceptors and visual function and mosttherapeutic interventions can at best only slow down the diseaseprogression.

Prior clinical studies in patients with retinal degeneration haveinvolved the use of fetal retinal sheet transplants. Thistransplantation strategy relies on the immature retinal sheet extendingcell processes and forming synaptic connections with the degenerate hostretina. The rationale behind this is that the inner retinal neurons ofthe host remain intact and therefore only require synaptic connectionswith photoreceptors for visual function to be restored. Studiesinvestigating retinal sheet transplantation in patients have shown somesubjective visual improvement, however graft rejection, tissueavailability, and unreliable clinical efficacy have prevented thisapproach from becoming a viable treatment option.

Stem cells and other pluripotent cells have also been contemplated foruse in treating patients with retinal degenerations and can be isolatedfrom a number of sources including embryonic tissue, adult brain,genetically manipulated dermal fibroblasts and even the retina. However,embryonic or stem cells have so far shown little ability todifferentiate into retinal phenotypes when transplanted into the adultretina unless first pre-differentiated into fetal-like retinalprogenitor populations. Moreover, the yields and efficiency ofengraftment are low and contamination with residual tumor-formingpluripotent cells has been problematic. The current challenge in thefield of photoreceptor cell placement involves understanding thedevelopmental processes that guide cells towards photoreceptordifferentiation, so that large numbers of these cells might betransplanted at the optimal stage with minimal risk of immune reactionsor transformation of implanted cells to a tumorigenic phenotype.

SUMMARY

In alternative embodiments, provided herein are compositions and methodsfor treating, ameliorating or preventing a retinal disease or condition,e.g., an Usher's disease, retinitis pigmentosa (RP), a degenerativeretinal disease, an age related macular degeneration (AMD), a wet AMD ora dry AMD, geographic atrophy, a retinal photoreceptor disease, adiabetic retinopathy, cystoid macular edema, uveitis, a retinaldetachment, a retinal injury, macular holes, macular telangiectasia, atraumatic or an iatrogenic retinal injury, a ganglion cell or opticnerve cell disease, a glaucoma or an optic neuropathy, an ischemicretinal disease such as retinopathy of prematurity, retinal vascularocclusion, or ischemic optic neuropathy; or improving a photopic (daylight) vision; or for improving correcting visual acuity, or improvingmacular function, or improving a visual field, or improving scotopic(night) vision. In alternative embodiments, provided herein areheterogeneous mixtures of fetal neural retinal cells and methods andcompositions (e.g., kits, formulations and the like) for making andusing them.

In alternative embodiments, provided herein are formulations, productsof manufacture or compositions comprising a heterogeneous mixture ofmammalian fetal neural retinal cells, made by a method comprising:

(a) harvesting a sample of cells comprising a plurality of mammalianfetal neural retinal cells from a mammalian fetus:

-   -   (i) at about 17 to 18 weeks gestational age, or at about 16 to        19 weeks gestational age, or 15 to 20 weeks, or 14 to 26 weeks,        or at about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26        weeks, gestational age for humans, or about 3, 4, 5, 6, 7, 8, 9,        10 or 11 weeks for non-human animals, optionally feline, canine        or porcine cells, or 6 to 7 for feline or 6 to 9 for porcine        cells, or    -   (ii) at a stage after which a mammalian retina is clearly formed        but before photoreceptor outer segments are fully formed and        retinal vascularization substantially completed or completed, or        at an analogous mammalian fetal staging,

wherein optionally the sample of cells comprise human or feline orcanine cells;

(b) enzymatically dissociating the harvested sample of cells to make adissociated suspension of cells and/or and small- and medium-sizedcellular clusters,

wherein optionally the harvested sample of cells and/or and smallcellular clusters are enzymatically dissociated using trypsin orequivalent, or _(TYRP-LE EXPRESS™) (TrypLE™ Express, Invitrogen-LifeTechnologies, Carlsbad Calif.) or equivalent; and

(c) culturing the cells and/or and small cellular clusters in a sterileenvironment comprising serum-free media or serum-comprising media, andantibiotics and antifungals or no antibiotics or anti-fungals, for nomore than about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more passages,

wherein optionally the cells and/or and small cellular clusters arecultured in a culture media comprising a Dulbecco's Modified EagleMedium: Nutrient Mixture F-12™ (DMEM/F12™) media or an ADVANCEDDMEM/F12™ media (Gibco-Invitrogen-Life Technologies, Carlsbad Calif.))or an ULTRACULTURE media (BioWhittaker-Lonza Walkersville, Inc.,Walkersville, Md.), optionally together with N2 supplement (Invitrogen)or B27 or B27 Xeno Free (Invitrogen), L-glutamine or GlutaMax(Invitrogen), and human recombinant growth factors consisting of EGF andbFGF (Invitrogen), or other growth factors,

and optionally the DMEM/F12™ media is used for human cells and theULTRACULTURE™ media is used for feline or canine cells,

and optionally culturing or growing the cells under low oxygenconditions, or oxygen conditions that approximate or closely mimicoxygen levels of a developing fetal retina during gestation, or at about2%, 2.5%, 3%, 3.5% oxygen,

and optionally the media is supplemented with vitamin C, and optionallythe vitamin C is added every 1 or 2 days, and optionally the vitamin Cis added in an amount to have an initial concentration of about 0.1mg/ml or 0.05 mg/ml, or between about 0.01 mg/ml to about 0.5 mg/ml),

and optionally the media is supplemented with albumin, or human orfeline or canine albumin, or recombinant albumin, or albumin is added inan amount to have an initial concentration of about 1.0 mg/ml),

and optionally the sample of cells is screened for the presence of apathogen, a bacteria, an endotoxin, a fungus, a mycoplasma, a virus, ahepatitis virus or an HIV virus,

and optionally the sample of cells is screened for the presence of anormal karyotype,

and optionally the sample of cells does not exhibit elevated telomeraseactivity,

and optionally the sample of cells is screened for viability,

optionally the sample of cells is screened for tumorigenicity.

In alternative embodiments of the formulations, products of manufactureor compositions, the method for making the heterogeneous mixture offetal neural retinal cells further comprises:

(a) selecting fetal neural retinal cells on the basis of cell surface orgenetic markers, optionally selecting the cells either before culturing(prospectively) or after culturing or both, wherein optionally the cellsurface or genetic markers comprise CD15/LeX/SSEA1 and/or GD2ganglioside; optionally CD9, CD81, CD133 or AQP4, CXCR4; or

(b) selecting fetal neural retinal cells on the basis of a fetal neuralretinal cell transcriptome profile, proteome profile and/or a genomicprofile.

In alternative embodiments, the method for making a heterogeneousmixture of fetal neural retinal cells of the invention furthercomprises:

culturing the cells under conditions that cause them to proliferate atan optimal rate (or near optimal rate) and/or express a fetal neuralretinal cell phenotype or transcriptome profile,

wherein optionally the fetal neural retinal cell phenotype profilecomprises expression of (optionally moderate) levels of Ki67, a p21,and/or a telomerase and/or high levels of one or more stem cell markersassociated with a multipotent but not a pluripotent cell,

or optionally the fetal neural retinal cell phenotype profile comprises(gene or message) expression of a nestin, a vimentin, a Ki-67, adifferentiation marker, a beta 3-tubulin, a glial fibrillary acidicprotein (GFAP) and/or a rhodopsin, or any combination thereof;

and optionally Dachl, Lhx2 and/or Pax6 messages are measured.

In alternative embodiments, the formulation, product of manufacture orcomposition is formulated for injection, or injection into a vitreouscavity or a subretinal space.

In alternative embodiments, provided herein are methods for treating aretinal disease or condition comprising:

(a) providing a formulation, product of manufacture or composition ofthe invention; and

(b) injecting the formulation, product of manufacture or composition of(a) into a vitreous cavity or a subretinal space,

wherein optionally the vitreous cavity or subretinal space is a human ora feline or canine vitreous cavity or subretinal space,

wherein optionally a standard intraocular injection procedure is used,

wherein optionally the method further comprises an anterior chamberparacentesis, thereby improving the safety of the procedure,

thereby treating the retinal disease or condition.

In alternative embodiments of the methods, the retinal disease orcondition comprises a Usher's disease, retinitis pigmentosa (RP), adegenerative retinal disease, an age related macular degeneration (AMD),a wet AMD or a dry AMD, a retinal photoreceptor disease, a diabeticretinopathy, a retinal detachment, a retinal injury, a traumatic or aniatrogenic retinal injury, a ganglion cell or optic nerve cell disease,a glaucoma or an optic neuropathy.

In alternative embodiments, provided herein are methods for improving aphotopic (day light) vision, comprising:

(a) providing a formulation, product of manufacture or composition ofthe invention; and

(b) injecting the formulation, product of manufacture or composition of(a) into a vitreous cavity or a subretinal space,

wherein optionally the vitreous cavity or subretinal space is a human ora feline or canine vitreous cavity or subretinal space,

wherein optionally a standard intraocular injection procedure is used,

wherein optionally the method further comprises an anterior chamberparacentesis,

thereby improving the photopic (day light) vision.

In alternative embodiments, provided herein are methods for improvingcorrecting visual acuity, or improving macular function, or improving avisual field, or improving scotopic (night) vision, comprising:

(a) providing a formulation, product of manufacture or composition ofthe invention; and

(b) injecting the formulation, product of manufacture or composition of(a) into a vitreous cavity or a subretinal space,

wherein optionally the vitreous cavity or subretinal space is a human ora feline or canine vitreous cavity or subretinal space,

wherein optionally a standard intraocular injection procedure is used,

wherein optionally the method further comprises an anterior chamberparacentesis,

thereby correcting visual acuity, or improving macular function, orimproving a visual field, or improving scotopic (night) vision.

In alternative embodiments, provided herein are methods for making aheterogeneous mixture of fetal neural retinal cells, made by a methodcomprising:

(a) at about 17 to 18 weeks gestational age, or at about 16 to 19 weeksgestational age, or 15 to 20 weeks, or 14 to 26 weeks, or at about 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 weeks, gestational agefor humans, or about 3, 4, 5, 6, 7, 8, 9, 10 or 11 weeks for non-humananimals, optionally feline, canine or porcine cells, or 6 to 7 forfeline or 6 to 9 for porcine cells,

wherein optionally the sample of cells comprise human or feline orcanine cells;

(b) enzymatically dissociating the harvested sample of cells to make adissociated suspension of cells and/or and small- and medium-sizedcellular clusters,

wherein optionally the harvested sample of cells are enzymaticallydissociated using trypsin or equivalent, or TRYP-LE EXPRESS™ (TrypLE™Express, Invitrogen-Life Technologies, Carlsbad Calif.) or equivalent;and

(c) culturing the cells in a sterile environment comprising serum-freemedia or serum-comprising media, and no antibiotics or anti-fungals, forno more than about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more passages,

wherein optionally the cells are cultured in a culture media comprisinga Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12™ (DMEM/F12™)media (Gibco-Invitrogen-Life Technologies, Carlsbad Calif.)) or anULTRACULTURE™ media (BioWhittaker-Lonza Walkersville, Inc.,Walkersville, Md.),

and optionally the DMEM/F12™ media is used for human cells and theULTRACULTURE™ media is used for feline or canine cells),

and optionally the media is supplemented with vitamin C, and optionallythe vitamin C is added every 1 or 2 days, and optionally the vitamin Cis added in an amount to have an initial concentration of about 0.1mg/ml or 0.05 mg/ml, or between about 0.01 mg/ml to about 0.5 mg/ml),

and optionally the media is supplemented with albumin, or human orfeline or canine albumin, or recombinant albumin, or albumin is added inan amount to have an initial concentration of about 1.0 mg/ml),

and optionally the sample of cells is screened for the presence of apathogen, a bacteria, an endotoxin, a fungus, a mycoplasma, a virus, ahepatitis virus or an HIV virus,

and optionally the sample of cells is screened for the presence of anormal karyotype,

and optionally the sample of cells does not exhibit elevated telomeraseactivity,

and optionally the sample of cells is screened for viability,

optionally the sample of cells is screened for tumorigenicity.

In alternative embodiments, provided herein are kits for treating aretinal disease or condition, or for practicing the method of theinvention, comprising:

(a) a formulation, product of manufacture or composition of theinvention; and/or

(b) a heterogeneous mixture of fetal neural retinal cells made by themethod of the invention.

In alternative embodiments, provided herein is a cell populationcomprising mammalian retinal progenitor cells expressing one or moremarkers selected from the group consisting of nestin, Sox2, Ki67, MHCClass I, and Fas/CD95, wherein nestin is expressed by greater than about90%, or about 95-99% of the cells in the population, wherein Sox2 isexpressed by greater than about 80%, or about 90-99% of the cells in thepopulation, wherein Ki-67 is expressed by greater than about 30%, orabout 40-60% of the cells in the population, wherein MHC Class I isexpressed by greater than about 70%, or about 90% of the cells in thepopulation, and wherein Fas/CD95 is expressed by greater than about 30%,or about 40-70% of the cells in the population. In some embodiments, thecells are derived from a human or non-human mammal.

In certain embodiments, the mammalian retinal progenitor cells in thepopulation further express one or more markers selected from the groupconsisting of vimentin, CD9, CD81, AQP4, CXCR4, CD15/LeX/SSEA1, GD2ganglioside, CD133, β3-tubulin MAP2, GFAP, OPN/SPP1, PTN, KDR, and TEK.

In another aspect, a method for isolating a population of mammalianretinal progenitor cells is provided, comprising harvesting mammalianfetal retinal tissue at a stage after which the retina is formed butbefore photoreceptor outer segments are fully formed throughout theretina and before retinal vascularization is substantially completed orcompleted; dissociating the harvested tissues to generate a dissociatedsuspension of cells and cell clusters; and culturing the dissociatedsuspension for about 10-30 passages, wherein the mammalian retinalprogenitor cells express one or more markers selected from the groupconsisting of nestin, Sox2, Ki-67, β3-tubulin, MAP2, MEW Class I, andFas/CD95, wherein nestin is expressed by greater than about 90%, orabout 95-99% of the cells in the population, wherein Sox2 is expressedby greater than about 80%, or 90-99% of the cells in the population,wherein Ki-67 is expressed by greater than about 30%, or about 40-60% ofthe cells in the population, wherein MEW Class I is expressed by greaterthan about 70%, or about 90% of the cells in the population, and whereinFas/CD95 is expressed by greater than about 30%, or about 40-70% of thecells in the population. In some embodiments, the tissues are harvestedfrom a human or a non-human mammal. In some embodiments, the tissues areharvested from a human fetal retina at a gestational age between about12 weeks to about 28 weeks, or from postnatal or neonatal retinaltissues. In other embodiments, the tissues are harvested from anon-human fetal retina at a gestational age between about 3 weeks toabout 11 weeks. In other embodiments, the tissues are harvested from anon-human mammal at a gestational age between about 3 weeks to 11 weeks,or from postnatal or neonatal retinal tissues. The cells may be culturedat atmospheric oxygen levels or at low oxygen levels that approximateoxygen levels of a developing fetal retina during gestation, such as,e.g., between about 0.5% to about 7% and may also be cultured inserum-free or reduced serum cell culture media, which may optionallycomprise additional supplements such as, for example, N2, B27, vitamin Cand albumin.

In other aspects, a method for treating a retinal disease or conditionin a subject in need thereof is provided, comprising administering tothe subject an effective amount of a composition comprising mammalianretinal progenitor cells, wherein the mammalian retinal progenitor cellsexpress one or more markers selected from the group consisting ofnestin, Sox2, Ki-67, β3-tubulin, MAP2, MEW Class I, and Fas/CD95,wherein nestin is expressed by greater than about 90%, or about 95-99%of the cells in the population, wherein Sox2 is expressed by greaterthan about 80%, or about 90-99% of the cells in the population, whereinKi-67 is expressed by greater than about 30%, or 40-60% of the cells inthe population, wherein MEW Class I is expressed by greater than about70%, or about 90% of the cells in the population, and wherein Fas/CD95is expressed by greater than about 30%, or about 40-70% of the cells inthe population, and optionally measuring changes in vision in thesubject, thereby treating the retinal disease or condition.

In alternative embodiments, the subject is a human or a non-humanmammal. In some embodiments, the composition is formulated for injectioninto a vitreous cavity or a subretinal space of the subject. The retinaldisease or condition may comprise retinitis pigmentosa (RP), Leber'scongenital amaurosis (LCA), Stargardt disease, Usher's syndrome,choroideremia, a rod-cone or cone-rod dystrophy, a ciliopathy, amitochondrial disorder, progressive retinal atrophy, a degenerativeretinal disease, age related macular degeneration (AMD), wet AMD, dryAMD, geographic atrophy, a familial or acquired maculopathy, a retinalphotoreceptor disease, a retinal pigment epithelial-based disease,diabetic retinopathy, cystoid macular edema, uveitis, retinaldetachment, traumatic retinal injury, iatrogenic retinal injury, macularholes, macular telangiectasia, a ganglion cell disease, an optic nervecell disease, glaucoma, optic neuropathy, ischemic retinal disease,retinopathy of prematurity, retinal vascular occlusion, familialmacroaneurysm, a retinal vascular disease, an ocular vascular diseases,a vascular disease, or ischemic optic neuropathy.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications cited herein are herebyexpressly incorporated by reference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following Detailed Description, given by way of example, but notintended to limit the subject matter described herein to specificembodiments described, may be understood in conjunction with theaccompanying figures, incorporated herein by reference, in which:

FIG. 1 illustrates the morphology of feline retina-derived progenitors,or RPCs. Morphology was maintained at different time points in thecourse of sustained culture.

FIG. 2 is a graph representing a growth curve showing the differences ingrowth of the feline RPCs in SM medium versus UL medium.

FIG. 3 shows the morphology of human RPCs (hRPCs) over time in culture(Day 0 to Day 56).

FIG. 4 illustrates a graph showing the growth kinetics of hRPCs overtime.

FIG. 5 illustrates a graph of a growth curve showing the growth kineticsof hRPCs over time.

FIGS. 6A-B graphically illustrate cell viability of human retinalprogenitor cells (hRPCs) as a function of growth conditions: FIG. 6Ashows comparison of growth conditions between Advanced DMEM/F12containing supplemental vitamin C and albumin, and base DMEM/F12 cellculture media supplemented with N2; FIG. 6B graphically illustrates anexperiment comparing growth of hRPCs in cell culture medium supplementedwith N2 or B27.

FIGS. 7A-C graphically illustrate cell viability of human retinalprogenitor cells (hRPCs) as a function of cell culture supplementationconditions: F 7A summarizes an experiment testing the effects of VitaminC supplementation in hRPC cultures of vitamin C resulted in improvedhRPC viability. FIG. 7B and FIG. 7C depict an experiment testing theeffects of albumin supplementation in hRPC cultures.

FIG. 8 reflects a comparison experiment testing differing osmolarity ofcell culture media on hRPC growth and viability.

FIG. 9A-C illustrates the results of experiments where hRPCs werecultured under conditions of atmospheric oxygen and compared to the samecells grown under low oxygen conditions; growth dynamics, morphology,and gene expression were evaluated, FIG. 9A graphically illustratesconfluence of cells under 3% versus 20% oxygen conditions; FIG. 9Billustrates an image of cells under 3% versus 20% oxygen conditions; andFIG. 9C graphically illustrates gene expression in various samples.

FIG. 10 graphically illustrates hRPC cell numbers under normoxia versushypoxia conditions, where hRPCs were cultured under conditions ofatmospheric oxygen and compared to the same cells grown under low oxygenconditions.

FIG. 11 graphically illustrates the reproducibility of growthcharacteristics for three different cell samples used to generate aworking cell bank, where hRPCs were cultured under conditions ofatmospheric oxygen and compared to the same cells grown under low oxygenconditions, and the graph shows hRPC cell numbers under normoxia versushypoxia conditions.

FIG. 12 shows the results of a steroid toxicity test performed on hRPCs.

FIGS. 13A-13D are graphs showing the viability and stability ofpreviously frozen hRPCs: FIG. 13A graphically illustrates hypoxic hRPCpost-thaw cell viability; FIG. 13B graphically illustrates normoxicpost-thaw cell viability; FIG. 13C graphically illustrates hypoxic andnormoxic post-thaw cell viability; FIG. 13D graphically illustrates agrowth curve as a function of cell numbers and passages in culture.

FIG. 14 shows feline RPCs stained by ImmunoCytoChemistry (ICC) markersnestin, β3-tubulin, vimentin, rhodopsin, Ki-67, and GFAP.

FIG. 15 is a graph showing a feline RPC gene profile over time usingqPCR of nestin, vimentin, GFAP, and PKC-α marker transcripts.

FIG. 16A is a graph illustration of differences in gene expression byqPCR measurement comparing feline RPC versus brain progenitor cells(BPCs).

FIG. 16B is a graph comparing feline RPC versus BPC.

FIG. 17 shows cell staining of RFP, vimentin, or opsin, ezrin or PKCafter in vivo transplantation of feline RPCs in the subretinal space ofdystrophic Abyssinian cats.

FIGS. 18A-F illustrate the morphology of human cells using markerexpression by ICC. The markers include: (A) nestin; (B) vimentin; (C)Sox2; (D) SSEA-1 (also known as CD15, LeX); (E) GD2-ganglioside; and (F)Ki-67;

FIGS. 19A-C illustrate the morphology of human cells using markerexpression by ICC. (A) shows β3-tubulin staining; (B) GFAP staining; and(C) GDNF staining.

FIG. 20 depicts marker expression at the RNA level, as detected byquantitative PCR heat mapping.

FIGS. 21A-21C show the results of a qPCR experiment comparing hRPCversus hFB and expanding marker detection: FIG. 21A shows the results ofqPCR (gene expression) analysis comparing expression levels of genes inhRPC versus human fibroblast (hFB). FIGS. 21B and 21C represent qPCR(gene expression) data from additional experiments.

FIG. 22 is a summary table showing a list of 11 genes (from the profileof approximately 26 used) that exhibit consistent behavior (in hRPC vshFB) between different donations.

FIG. 23 shows marker expression at RNA level, as detected by qPCR (“realtime” PCR) at time points in culture.

FIG. 24 depicts expression level changes of various markers (e.g., GDNF,annexin V, β3-tubulin, Notchl, Six6, Ki-67, and CD133 in early vs. latepassage cells.

FIGS. 25A-E represents a summary of microarray data that distinguisheshRPCs from neural stem cells (BPCs).

FIG. 26 shows marker expression after differentiation with retinoic acid(RA).

FIG. 27 is a summary table of culture conditions of hRPCs grown fromdifferent tissue donations.

FIGS. 28A-B illustrate differences in gene expression between hRPCsderived from different donors and cultured in a variety of cell culturemedia conditions and time points were tested: FIG. 28A illustrates geneexpression by qPCR grown in standard proliferation medium (SM)conditions at different time points in culture; FIG. 28B illustratesgene expression by qPCR grown in SM-UL (initial UL, then SM) conditionsat two different time points.

FIGS. 29A-C illustrates differences in gene expression between hRPCsderived from different donors and cultured in a variety of cell culturemedia conditions and time points were tested: FIG. 29A illustrates geneexpression by qPCR in standard proliferation medium (SM)-FBS (initialplating conditions SM+5% FBS, then changed to SM alone) conditions atdifferent time points. FIG. 29B illustrates gene expression by qPCR withSM alone, at 2 different time points. FIG. 29C illustrates geneexpression by qPCR with SM-hS (SM, after initial SM+human serum) at thesame 2 time points. As previously, only GDNF is elevated.

FIG. 30 is a table summary of time point comparison experiment resultsshowing FIGS. 28A-B and FIGS. 29A-29C. Genes showing strongly consistenttrends across treatment conditions are highlighted in yellow.

FIG. 31 depicts a heat map analysis of qPCR data obtained from hRPCs.

FIGS. 32A-C represents a histogram of qPCR data from hRPCs.

FIGS. 33A-B depicts qPCR expression levels of angiogenesis pathwayangiogenesis-related genes in hRPCs. FIG. 33B depicts qPCR expressionlevels of WNT-pathway related genes in hRPCs.

FIGS. 34A-C is a graph summarizing the results of Principal ComponentAnalysis (PCA) showing differences in qPCR gene expression levelsbetween hRPCs vs. tissue and fibroblasts. FIG. 34B is another PCA graphshowing differences in qPCR gene expression between hRPCs vs. tissuesobtained from fetal retina at day 0. FIG. 34C is another PCA graphdepicting three-dimensional visualization of the global similarities anddifferences between cell sample populations, lines are drawn to separateneural retina and neural retina-derived cells (left) from non-neuralretinal cells (upper right).

FIG. 35 shows cluster analysis of three hRPC populations (hypoxic MCB,normoxic MCB, and hypoxic WCB) versus fetal retinal tissue.

FIG. 36 is a Volcano plot comparing differences in gene expression inhypoxic MCB vs. tissue.

FIGS. 37A-B is a Venn diagram showing the number of differentiallyexpressed genes between hRPC groups as a function of the treatmentconditions hypoxia MCB, hypoxia WCB and normoxia, with fetal retinaltissue used as comparator. FIG. 37B is a Venn diagram showing the numberof differentially expressed genes between hRPC groups normalized totissue, as a function of passage number and treatment conditions hypoxiaMCB and hypoxia WCB.

FIGS. 38A-B graphically illustrate, or plot, each gene as a data point,plotted relative to fold change (up or down, X axis) and statisticalsignificance (function of p-value, Y axis), providing an overview of howmany genes are changing, how much and in which direction (up vs. down),called Volcano plots, comparing different hRPCs vs. tissue of origin.

FIGS. 39A-B graphically illustrate, or plot, each gene as a data point,plotted relative to fold change (up or down, X axis) and statisticalsignificance (function of p-value, Y axis), providing an overview of howmany genes are changing, how much and in which direction (up vs. down),called Volcano plots comparing hypoxic hRPCs vs. normoxic hRPCs.

FIG. 40 is a table summarizing time point design for feline RPCs: tableof donations and treatment conditions are compared (the red underscoreat UL-d76 corresponds to a clear upward inflection in growth curve forthe cells, i.e., possible spontaneous immortalization).

FIG. 41A-B show the results of an experiment measuring gene expressionlevels in feline RPCs by qPCR in UL media at the various time points:FIG. 41A illustrates gene expression by qPCR of feline cRPCs grown in ULmedium at indicated time points; and FIG. 41B includes additionalmarkers not shown in FIG. 41A.

FIG. 42A-B illustrate additional data from the same experiment as shownin FIGS. 41A-B: FIG. 42A illustrates cRPC-UL time points based on geneexpression; and, FIG. 42B, a continuation of FIG. 42A, illustratescRPC-UL time points based on gene expression.

FIG. 43A-B illustrate an experiment measuring gene expression levels infeline RPCs by qPCR in UL media at the various time points: FIG. 43Aillustrates cRPC-SM time points based on gene expression; and, FIG. 43B,a continuation of FIG. 43A, illustrates cRPC-SM time points based ongene expression.

FIG. 44 illustrates rcRPC-UL time points based on gene expression.

FIG. 45A-B is a radial graph showing changes in the expression patternof feline cRPCs by time point and culture conditions: FIG. 45Aillustrates cRPC time points for d0, d31 SM and d31 UL; and, FIG. 45Billustrates cRPC time points for d0, d31 SM, d31 UL, d52 UL, d97 UL andd114 IL.

FIG. 46 is a chart summarizing qPCR data obtained from feline RPCsacross different donations and culture conditions.

FIG. 47 shows the results of an ELISA test characterizing hRPCs culturedunder normoxic and hypoxic conditions. Markers detected include OPN,VEGF, SDF-1, BDNF, and GDNF.

FIG. 48 show the results of a FACS analysis of hRPCs cultured undernormoxic and hypoxic conditions. Markers of interest include nestin,Sox2, Ki-67, GFAP, MHC Class I and II, Fas/CD95, CXCR4, CD15, and GD2ganglioside.

FIG. 49A-E illustrates proof of concept of methods as provided hereinusing in vivo transplantation of hRPCs into the eyes of dystrophic RCSrats in a model of a hereditary photoreceptor degeneration: FIG. 49Agraphically illustrates an optomoter response for P60;

FIG. 49B graphically illustrates an optomoter response for P90; FIG. 49Cgraphically illustrates the luminance threshold response for P90; FIG.49D illustrates an image of the histology of a P60 intravitrealinjection; FIG. 49E illustrates an image of the histology of a P90intravitreal injection.

FIG. 50A-B illustrate photographs showing staining of rat retinas aftersubretinal injection of hRPCs: FIG. 50A illustrates an image of a retinaafter subretinal injection of hRPCs, the section of retina isillustrated in FIG. 50C; FIG. 50B illustrates an image of a retina awayfrom the injection site, the section of retina is illustrated in FIG.50C; and, FIG. 50C illustrated the sections of the retina taken for FIG.50A and FIG. 50B.

FIG. 51A-C illustrate photographs showing staining of rat retinas aftersubretinal injection of hRPCs: FIG. 51A illustrates an image of a retinaafter subretinal injection of vehicle only, the section of retina isillustrated in FIG. 51C; FIG. 51B illustrates an image of a retina awayfrom the injection site, the section of retina is illustrated in FIG.51C; and, FIG. 51C illustrates the sections of the retina taken for FIG.51A and FIG. 51B.

FIG. 52A-B illustrate photographs showing staining of rat retinas afterintravitreal injection of hRPCs: FIG. 52A illustrates an image of aretina after intravitreal injection of hRPCs, the section of retina isillustrated in FIG. 52C; FIG. 52B illustrates an image of a retina awayfrom the injection site, the section of retina is illustrated in FIG.52C; and, FIG. 52C illustrates the sections of the retina taken for FIG.52A and FIG. 52B.

FIG. 53A-B illustrate photographs showing staining of rat retinas afterintravitreal injection of hRPCs: FIG. 53A illustrates an image of aretina after intravitreal injection of vehicle only, the section ofretina is illustrated in FIG. 53C; FIG. 53B illustrates an image of aretina away from the injection site, the section of retina isillustrated in FIG. 53C; and, FIG. 53C illustrates the sections of theretina taken for FIG. 53A and FIG. 53B.

FIG. 54A-D are photographs showing immunocytochemical staining of RCSwhole mounts after injection of hRPCs in rat retinas. FIG. 54A and FIG.54B: sham; FIG. 54C and FIG. 54D: treated.

FIG. 55 illustrates a graph showing improvements in visual acuity inpatients receiving treatment with hRPCs.

DETAILED DESCRIPTION

The features, structures, or characteristics described throughout thisspecification may be combined in any suitable manner in one or moreembodiments. For example, the usage of the phrases “exemplaryembodiments,” “example embodiments,” “some embodiments,” or othersimilar language, throughout this specification refers to the fact thata particular feature, structure, or characteristic described inconnection with an embodiment may be included in at least one embodimentdescribed herein. Thus, appearances of the phrases “exemplaryembodiments,” “example embodiments,” “in some embodiments,” “in otherembodiments,” or other similar language, throughout this specificationdo not necessarily all refer to the same group of embodiments, and thedescribed features, structures, or characteristics can be combined inany suitable manner in one or more embodiments.

To facilitate the understanding of this disclosure, a number of termsare defined below. Terms defined herein have meanings as commonlyunderstood by a person of ordinary skill in the areas relevant to thesubject matter described herein. Terms such as “a”, “an” and “the” arenot intended to refer to only a singular entity, but include the generalclass of which a specific example may be used for illustration. Theterminology herein is used to describe specific embodiments of thesubject matter described herein, but their usage does not delimit thesubject matter, except as outlined in the claims.

In alternative embodiments, provided herein are compositions and methodscomprising or using heterogeneous mixtures of fetal neural retinal cellsfor treating, ameliorating or preventing a retinal disease or condition,e.g., Usher's disease, retinitis pigmentosa (RP), a degenerative retinaldisease, an age related macular degeneration (AMD), a wet AMD or a dryAMD, a retinal photoreceptor disease, a diabetic retinopathy, a retinaldetachment, a retinal injury, a traumatic or an iatrogenic retinalinjury, a ganglion cell or optic nerve cell disease, a glaucoma or anoptic neuropathy; or improving a photopic (day light) vision; or forimproving correcting visual acuity, or improving macular function, orimproving a visual field, or improving scotopic (night) vision. Inalternative embodiments, this invention provides heterogeneous mixturesof fetal neural retinal cells and methods and compositions (e.g., kits,formulations and the like) for making and using them.

In alternative embodiments, provided herein are methods and uses ofcultured retinal progenitor cells prepared as a cell suspension and usedas allogeneic grafts injected into the vitreous cavity of patients withretinal disease. In alternative embodiments, provided herein arecell-based therapies comprising or consisting of use of culturedheterogeneous cell populations from an immature mammalian, e.g., humanretina.

In alternative embodiments, proliferating mammalian, e.g., human retinalcells are grown from donor tissue, characterization performed, and cellsinjected under topical anesthesia directly to the vitreous cavity,without need for systemic immune suppression of the recipient, e.g., apatient.

In alternative embodiments, the compositions and methods of theinvention are used for the treatment, prevention or amelioration of aretinal disease, e.g., a retinal degeneration, e.g., a retinitispigmentosa (RP).

While the invention is not limited by any particular mechanism ofaction, in one embodiment, donor fetal retinal cells provide a trophicinfluence for the host retina, notably including host cones. Thistrophic effect is not only neuroprotective, but also has a rapidrevitalizing effect on residual host retinal cells as determined byimproved visual function. In one embodiment, donor cells are capable ofintegrating into the retina and, via cellular differentiation, replacephotoreceptors (which can be in limited numbers). The overall effect isto both rapidly and sustainably restore and preserve clinicallysignificant degrees of visual function in a retina otherwise destined tofail completely, leaving the patient completely blind. Accordingly, inone embodiment, the compositions and methods of the invention canrapidly and sustainably restore and preserve clinically significantdegrees of visual function in a retina in a mammal, e.g., a human.

In alternative embodiments, methods of the invention comprise making andusing dissociated suspensions of fetal retinal cells, e.g., humanretinal progenitor cells (hRPCs), and optionally not including tissue orscaffolds. In alternative embodiments, these cells are injected in avitreous cavity; where optionally no vitrectomy or subretinal surgery isrequired. In one embodiment, cells can be efficaciously implanted into(e.g., injected into) a subretinal space, or, they can be efficaciouslyimplanted into (e.g., injected into) an eye using any standardintraocular injection procedure, e.g., using a hypodermic or an angledinsertion pathway. In alternative embodiments no retinotomy or nointraocular gas or silicon oil is required.

In alternative embodiments, an anterior chamber paracentesis also can beperformed, or not depending on situation, as determined by one of skillin the art. In alternative embodiments, no suturing of globe is neededduring and/or after a procedure. In alternative embodiments only topicalanesthesia is used, e.g., no local, regional, general anesthesia used.

In alternative embodiments, no anti-inflammatory and/or immunesuppression is used; but optionally anti-inflammatory and/or immunesuppression therapy as post-operative drops can be included. Inalternative embodiments, tissue typing of graft and matching to patientis not required.

In alternative embodiments, there is no mandatory bed rest, post-opand/or need for “face-down” positioning. In alternative embodiments, amethod of the invention is performed an outpatient procedure, andoptionally does not require any overnight hospital stay.

In alternative embodiments, compositions and methods of the inventionare used to prevent, ameliorate and/or treat a retinitis pigmentosa(RP), Usher's disease, or any degenerative retinal disease, e.g., AMD,or a retinal photoreceptor disease such as a retinal detachment, or aretinal disease such as a diabetic retinopathy, or a ganglion cell/opticnerve disease such as glaucoma or optic neuropathy.

In alternative embodiments, using or practicing the compositions andmethods of the invention results in: improved photopic (day light)vision, optionally providing a rapid effect;

increased best-corrected visual acuity; improved macular function,possibility of preserving or regaining central fixation; improved visualfield; improvements in scotopic (night) vision, with time; where thereis a concomitant hearing loss an in Usher's Syndrome, a reportedincreased (improved) sensitivity to sound; and/or for patients with LPvision (light perception only), where a treated eye has limitedimprovement—a marked improvement in visual acuity in a contralateral eyeis provided.

In alternative embodiments, using or practicing the compositions andmethods of the invention results in various systemic benefits, e.g.,changes in appearance in treated individuals possibly due to somaticimprovements, which could be related to effect of light on circadianrhythms, pituitary function, release of hormones, vascular tone, etc.;improved sense of visual capabilities; improved ambulatory independence;improved sense of well-being; and/or improved activities of dailyliving.

In alternative embodiments, using or practicing the compositions andmethods of the invention does not result in: development of unwantedcell growth, e.g., tumors; infections, e.g., no endopthalmitis—a riskfor any intraocular procedure; transmission of disease (however, prionor mad cow disease may be difficult to rule out; uveitis and/or acutegraft rejection; elevated intraocular pressure; angle closure; hypotony;retinal detachment and/or neovascularization.

In alternative embodiments, compositions and methods of the inventionuse retinal progenitor cultures comprising or consisting of:heterogeneous cultures of immature retinal cells, obtained from a fetalmammalian retina, which optionally are not clonally selected, butoptionally are mixed. In alternative embodiments, the cells expressprogenitor markers and retinal markers. In alternative embodiments, thecells are raised under xeno-free conditions for clinical use, sincexeno-contamination can be a safety issue. In alternative embodiments thecells are grown under completely serum-free conditions, or, the cellscan be grown in a serum-containing condition, if desired.

In alternative embodiments the cells are not immortal, nor are theyallowed to immortalize, or forced to immortalize. In alternativeembodiments the cells do not proliferate indefinitely. Exemplary cellculture methods of this invention can improve rate and duration, and canimprove donor cell yield significantly for a given tissue donation.

In alternative embodiments the cells are not stem cells per se, butrather they are immature and/or plastic, and optionally do not meet thedefinition for true stem cells.

In alternative embodiments, cells used to practice the invention areobtained from mammalian fetal tissue, and optionally do not persist forthe life of the organism. In alternative embodiments, cell used topractice the invention cannot (in the absence of additionalmanipulation) give rise to a germ layer, or cannot (in the absence ofadditional manipulation) give rise to all three (3) germ layers;optionally they are pre-specified to make retinal tissue or cells.

In alternative embodiments, cells used to practice the invention are apopulation of closely related cells, not an isolated single cell type.In alternative embodiments, cell used to practice the invention are notpluripotent, and optionally can appear to be multipotent. In alternativeembodiments, cells used to practice the invention have never beencultured in a pluripotent state; therefore they are safer. Inalternative embodiments, cells used to practice the invention are notgenetically modified, or alternatively, they are genetically modified(e.g., transformed stably or transiently, or inducibly).

In alternative embodiments, compositions and methods of the inventionprovide clinically significant trophic influences to a diseased retina,or provide regenerative influences to a macular and/or a scotopic visualfunction.

In alternative embodiments, cells used to practice the invention havelow immunogenicity, e.g., as allografts, when placed in the eye, or in avitreous cavity of the eye, or in a subretinal space.

In alternative embodiments, cell used to practice the invention areretinal progenitor cells (RPCs), as distinguished from a neuralprogenitor and/or a neural stem cells (NSCs). In alternativeembodiments, mammalian fetal retinal or RPC cells used to practice theinvention are multipotent, but are not equivalent to NSCs. Inalternative embodiments, mammalian fetal retinal or RPC cells used topractice the invention are not from the brain, but are from the retina.In alternative embodiments, mammalian fetal retinal or RPC cells used topractice the invention give rise to photoreceptors (brain-derivedprogenitors are poor at this). In alternative embodiments, mammalianfetal retinal or RPC cells used to practice the invention aremultipotent, but do not (in the absence of additional manipulation) giverise to oligodendrocytes (unlike NSCs).

In alternative embodiments, mammalian fetal retinal or RPC cells used topractice the invention are express quantitatively different geneprofiles, e.g., as described herein; or they express quantitativelydifferent soluble factor profiles; or they express quantitativelydifferent surface marker profiles.

In alternative embodiments, mammalian fetal retinal or RPC cells used topractice the invention are from a mammalian fetal neural retina, not aciliary margin, ciliary epithelium, nor RPE. In alternative embodiments,mammalian fetal retinal or RPC cells used to practice the invention arenot descended from differentiated Mueller glia, they are notpost-mitotic precursors per se, are not stem cells per se, and/or arenot a single isolated cell type per se.

In alternative embodiments, mammalian fetal retinal cells or RPC cellsused to practice the invention have a gene profile that is not fixed,constant or immutable; or they have a gene profile that dynamicallychanges quantitatively with time in culture.

In alternative embodiments, mammalian fetal retinal cells or RPC cellsused to practice the invention are not found in the early embryo, e.g.,the blastocyst. In alternative embodiments, mammalian fetal retinalcells or RPC cells used to practice the invention are not found in anyuseful abundance in the normal mature mammal, e.g., human. Inalternative embodiments, mammalian fetal retinal cells or RPC cells usedto practice the invention are found in their native abundance in thedeveloping (fetal) mammalian, e.g., human, retina. In alternativeembodiments, mammalian fetal retinal cells or RPC cells used to practicethe invention do not normally reside in the bone marrow, nor are derivedfrom same.

In alternative embodiments, mammalian fetal retinal cells or RPC cellsused to practice the invention are mostly mitotic when grown underproliferation conditions, optionally along with a minority admixture ofpost-mitotic cells. In alternative embodiments, mammalian fetal retinalcells or RPC cells used to practice the invention are derivedartificially from pluripotent cell lines, although optionally containingno population of residual pluripotent cell types.

In alternative embodiments, mammalian fetal retinal cells or RPC cellsused to practice the invention give rise to (differentiate into) retinalcells including photoreceptors, but not oligodendrocytes.

In alternative embodiments, mammalian fetal retinal cells or RPC cellsused to practice the invention are immunologically tolerated as ocularallografts in unrelated mammals, e.g., humans. In alternativeembodiments, mammalian fetal retinal cells or RPC cells used to practicethe invention are grafted to a vitreous cavity for mammalian, or human,vision or retinal disease therapeutic or prophylactic therapy.

In alternative embodiments, mammalian fetal retinal cells or RPC cellsused to practice the invention do not come with any, or a substantialrisk, of tumor formation or other unwanted cell growth.

In alternative embodiments, mammalian fetal retinal cells or RPC cellsused to practice the invention are cultured as spheres or adherentmonolayers, or as spheres and then monolayers, or as a combination ofspheres and monolayers. In alternative embodiments, spheres are notrequired, or the cells are grafted as dissociated cells, not as spheres,or as mixture of both. In alternative embodiments, mammalian fetalretinal cells or RPC cells used to practice the invention comprisegrafted cells that coalesce in vitreous, and optionally can becomespheres.

While the invention is not limited by any particular mechanism ofaction, an exemplary mechanism of action is diffusible and/or trophic;evidence is consistent with concept of trophic reprogramming of moribundhost cones, resulting in switch from apoptotic trajectory toregeneration of photic processing capability. This can be direct orindirect. Involvement of other ocular tissues not ruled out. Thismechanism allows for placement of a graft of a heterogeneous mixture offetal neural retinal cells of the invention in a vitreous or asubretinal space. In one aspect, vitreal placement enhancesdiffusion-based treatment effect. This mechanism can allow a graft ofheterogeneous mixtures of fetal neural retinal cells of the invention tobe placed out of the visual axis, yet still treat patient's macula.

In alternative embodiments, a vitreal placement is used to greatlysimplify a treatment. This exemplary treatment can increase availabilityto needy patients worldwide; vitreal placement may aid in immunetolerance by being remote to vasculature. While the invention is notlimited by any particular mechanism of action, in a relatively cell-freespace, with few native antigen presenting cells, the exemplary vitrealplacement of the invention: avoids potential complications of subretinalsurgery, avoids risks of general anesthesia, does not require that ahole (retinotomy) be made in retina (which raises risk of retinaldetachment, bleeding), and/or does not require the patient's retinaundergo a focal detachment (focal detachment can lead to tears,bleeding, global detachment; in RP, detachment of diseased retinal willbe a difficult/risky procedure).

In alternative embodiments visual benefits are rapid and may occurwithin first week post-treatment. In alternative embodiments,incremental benefits occur over longer periods.

In alternative embodiments, retinal cell replacement is possible, butnot required for clinical efficacy; donor cell migration into retina ispossible, but not required for clinical efficacy. In alternativeembodiments, donor cell integration in retinal circuitry is possible,but not required for efficacy; donor cell integration into the outernuclear layer/macula of host retina is possible, but not required forefficacy. In alternative embodiments, donor cell integration into retinais possible but not required for sustained graft survival.

In alternative embodiments, donor cells are cultured withoutantibiotics; this can avoid altering cells; and without antibiotics useof very low passage cells is possible since occult microbialcontamination can be ruled out. In alternative embodiments, use of lowpassage cells have a low risk of transformation and/or tumor formation;and, use of low passage cells are closest to the natural cells presentin the developing retina.

In alternative embodiments, DMEM/F12-based media or equivalents arepreferential for growing human RPCs; and Ultraculture-based media orequivalents are preferential for growing feline progenitors.

The subject matter disclosed herein relates to a cell populationcomprising mammalian retinal progenitor cells that are isolatedaccording to a defined cell culture method and which expresscharacteristic markers. The cell population may be a culture of cellsisolated from a mammal and grown in vitro. For example, the culture maycomprise a suspension of cells or adherent cells cultured in a cultureplate, dish, flask, or bioreactor. The sample may be homogeneous orheterogeneous, which may be determined by expression of one or moremarkers as defined herein. In some embodiments, the cell populationdisclosed herein is a mixed cell population and may contain a mixture ofundifferentiated and differentiated cells. Relative expression levels ofmarkers characteristic of the retinal progenitor cells defined hereinmay vary between cells within the population.

In alternative embodiments, the term “purified” or “enriched” indicatesthat the cells or cell populations are removed from their normal tissueenvironment and are present at a higher concentration as compared to thenormal tissue environment. Accordingly, a “purified” or “enriched” cellpopulation may further include cell types in addition to retinalprogenitor cells and may include additional tissue components, and theterm “purified” or “enriched” does not necessarily indicate the presenceof only progenitor cells or exclude the presence of other cell types.

In alternative embodiments, the retinal progenitor cell populations asdisclosed herein may be at least 5% pure, at least 10% pure, at least15% pure, at least 20% pure, least 25% pure, at least 30% pure, at least35% pure, at least 40% pure, at least 45% pure, at least 50% pure, atleast 55% pure, at least 60% pure, at least 65% pure, at least 70% pure,at least 75% pure, at least 80% pure, at least 85% pure, at least 90%pure, at least 95% pure, at least 96% pure, at least 97% pure, at least98% pure, at least 99% pure or at any increment between 5% and 99% pure.

In alternative embodiments, a “marker” refers to any molecule that canbe observed or detected. For example, a marker can include, but is notlimited to, a nucleic acid, such as a transcript of a specific gene, apolypeptide product of a gene, a non-gene product polypeptide, aglycoprotein, a carbohydrate, a glycolipid, a lipid, a lipoprotein or asmall molecule (for example, molecules having a molecular weight of lessthan 10,000 Daltons). In alternative embodiments, retinal progenitorcells may be characterized by the presence of one or more markers thatcan be expressed on the surface of the cells within the cell population(a “cell surface marker”), inside cells within the cell population(i.e., in the nucleus or cytoplasm of a cell), and/or expressed at theRNA or protein level as a “genetic” marker.

In alternative embodiments, the terms “express” and “expression” as usedherein refers to transcription and/or translation of a nucleic acidsequence within a host cell. The level of expression of a desiredproduct/protein of interest in a host cell may be determined or“screened” on the basis of either the amount of corresponding mRNA thatis present in the cell, or the amount of the desired polypeptide/proteinof interest encoded by the selected sequence as in the present examples.For example, mRNA transcribed from a selected sequence can be quantifiedor detected by Northern blot hybridization, ribonuclease RNA protection,in situ hybridization to cellular RNA, microarray analysis, or byreverse-transcription polymerase chain reaction (RT-PCR). Proteinsencoded by a selected sequence can be detected or quantified by variousantibody-based methods, e.g. by ELISA, by Western blotting, byradioimmunoassays, by immunoprecipitation, by assaying for thebiological activity of the protein, by immunostaining of the protein(including, e.g., immunohistochemistry and immunocytochemistry), by flowcytometry or fluorescence activated cell sorting (“FACS”) analysis, orby homogeneous time-resolved fluorescence (HTRF) assays.

Retinal progenitor cells may be characterized by their expression ofmolecular markers, including cell surface markers and non-surface(“genetic”) markers. While it is common in the art to refer to cells as“positive” or “negative” for a particular marker, actual expressionlevels are a quantitatively determined. The number of molecules on thecell surface (or located elsewhere) can vary by several logs, yet stillbe characterized as “positive”. It is also understood by those of skillin the art that a cell which is negative for staining, i.e. the level ofbinding of a marker specific reagent is not detectably different from acontrol, e.g. an isotype matched control, may express minor amounts ofthe marker. Characterization of the level of labeling (“staining”)permits subtle distinctions between cell populations. The stainingintensity of cells can be monitored by flow cytometry, where lasersdetect the quantitative levels of fluorochrome (which is proportional tothe amount of cell surface marker bound by specific reagents, e.g.antibodies). Flow cytometry, or FACS, can also be used to separate cellpopulations based on the intensity of binding to a specific reagent, aswell as other parameters such as cell size and light scatter. Althoughthe absolute level of staining may differ with a particular fluorochromeand reagent preparation, the data can be normalized to a control.

To normalize the distribution to a control, each cell is recorded as adata point having a particular intensity of staining. These data pointsmay be displayed according to a log scale, where the unit of measure isarbitrary staining intensity. By way of example, the brightest stainedcells in a sample can be as much as 4 logs more intense than unstainedcells. When displayed in this manner, cells falling in the highest logof staining intensity are bright, while those in the lowest intensityare negative. The “low” positively stained cells have a level ofstaining above the brightness of an isotype matched control, but are notas intense as the most brightly staining cells normally found in thepopulation. Low positive cells may have unique properties that differfrom the negative and brightly stained positive cells of the sample. Analternative control may utilize a substrate having a defined density ofmarker on its surface, for example a fabricated bead or cell line, whichprovides the positive control for intensity.

Expression of markers may be subject to change during culture of retinaltissue from which the retinal progenitor cells and cell populations arederived. For example, differences in marker expression can be influencedby culture conditions such as oxygen levels (i.e., atmospheric oxygenconditions, or “normoxic” conditions; or low oxygen conditions, alsoknown as “hypoxic” conditions). Those of ordinary skill in the art willbe aware that marker expression of the retinal progenitor cells and cellpopulations is not static and may change as a function of one or moreculture conditions, i.e., culture media, oxygen levels, number ofpassages, time in culture, etc.

Retinal progenitor cells and cell populations may express one or more,two or more, three or more, four or more, five or more, six or more,seven or more, eight or more, nine or more, ten or more, eleven or more,twelve or more, thirteen or more, fifteen or more, sixteen or more,seventeen or more, eighteen or more, nineteen or more, twenty or more,twenty-five or more, thirty or more of the markers defined herein, orany increment in between up to fifty or more markers.

In alternative embodiments retinal progenitor cells and cell populationscomprising retinal progenitor cells are characterized or screened byexpression of one or more markers such as, e.g., nestin, vimentin, Sox2,Ki67, MHC Class I, Fas/CD95, MAP2, CD9, CD81, AQP4, CXCR4,CD15/LeX/SSEA1, GD2 ganglioside, CD133, β3-tubulin, GFAP, OPN/SPP1, PTN,KDR, and TEK. In certain embodiments, the retinal progenitor cells andcell populations express one or more markers selected from the groupconsisting of nestin, Sox2, Ki-67, MHC Class I, and Fas/CD95, whereinnestin is expressed by greater than 90%, or 95-99% of the cells in thepopulation, wherein Sox2 is expressed by greater than 80%, or 90-99% ofthe cells in the population, wherein Ki-67 is expressed by greater than30%, or 40-60%) of the cells in the population (i.e., 60-85% of cellsgrown under normoxic/atmospheric oxygen conditions, 80-90% of cellsgrown under hypoxic conditions), wherein MHC Class I is expressed bygreater than 70%, or 90% of the cells in the population, and whereinFas/CD95 is expressed by greater than 30%, or 40-70% of the cells in thepopulation. In some embodiments, the retinal progenitor cells and cellpopulations further express one or more markers selected from the groupconsisting of vimentin, CD9, CD81, AQP4, CXCR4, CD15/LexA/SSEA1, GD2ganglioside, CD133, β3-tubulin, MAP2, GFAP, OPN/SPP1, PTN, KDR, and TEK.GFAP may be expressed by 5-10% of the cells in the population. MHC ClassII may be expressed by 1-3% of the cells in the population. CXCR4 may beexpressed by 5-30% of cells grown under atmospheric oxygen conditions,but 90% of cells grown under hypoxic conditions. CD15 may be expressedby 4-35% of cells in the population, i.e., 4-8% of cells grown underatmospheric oxygen conditions, at 15-35% of cells grown under hypoxicconditions. GD2 may be expressed by 2-15% of cells in the population,i.e., 2-4% of cells grown under atmospheric oxygen conditions, 15% ofcells grown under hypoxic conditions.

In alternative embodiments retinal progenitor cells and cell populationsare characterized or screened for low, trace, negative, or decreasedexpression of one or more of ABCA4, AIPL1, AKT3, APC2, BSN, CCNG2CDHR1,CRX, CD24, Claudin 11, CNTF, CNTFR, DACH1, DAPL1, DCX, DLG2 and 4, DLL4,EPHA7, EYS,FLT1, FSTL5, FZD5, FGF9, 10, and 14, GADD45G, GRIA2, HES5 and6, HEY2, HEYL, HGF, HIF3A, IMPG1 and 2, JAK2 and 3, KLF4, MAP6, myelinbasic protein (MBP), MYCN, Nanog, NBL1, NEFL, NEFM, NEUROD1, NEUROD4,NEUROG1, NEUROG2, NOTCH 1, 2, and 3, NRL, NRCAM, NRSN, NRXN1, 2, and 3,OCT4, OLIG2, OPN1MW1 and 2, OPN1SW, OTX2,

PAR4, PAX6, PRPH2, RAX1 and 2, RBP3, RCVRN, RELN, RGR, rhodopsin,RICTOR, RP1, RRH, RXRG, SIX3 and 6, SOX 8, SLC25A27, STAT1, STAT3, SYP,SYT4, WIF1, VSX2, and VSX1 and 2. The expression pattern of thesemarkers may be used to distinguish retinal progenitor cells or cellpopulations from tissues of origin, e.g., freshly isolated retinaltissues.

In alternative embodiments other markers whose expression may beincreased relative to tissues of origin include, without limitation,ADM, ANGPT1, ANGPTL2 and 4, ATP5D, BHLHE40, CCL2, CCNB1, CCND2, andCCNDE1, CD44, CDKN2A, Claudin 1, 4, and 6, CPA4, CTGF, CXCL12, DKK2,EMP1, FOXC2, FZD6, GADD45B, HES1, HIF1A, HOXB4, IGF1, IGFBP3, 5, and 7,IL1A,IL1R, IL1RAP, IL4R,IL7R, IL11, IL18, JAG1, LIF, LOX, BDNF, EGF,EGFR, FGF1, 2, 5, and 9, KLF4, 5 and 12, MITF, MMP1, MYC, NCAN, NEFH,NOG, NTF3, NTRK2, NRP1 and 2, OSMR (IL31RA), OTX1, PAX8, PDGFA, B, andC, PLAU, PRRX1, RPE65, SDF-1, SFRP1 and 4, SIX1 and 4, SLC25A1, 19, and20, TEK/TIE1, THBS1 and 2, TLR4, VEGFA, VEGFC WNT5A, and WNT7B.

In alternative embodiments retinal progenitor cells and cell populationsare distinguished from other central nervous system progenitor celltypes like brain progenitor cells and neural stem cells, and otherswhich may be derived from fetal CNS tissues such as brain and spinalcord. Markers that may be increased relative to other CNS progenitorcells include, without limitation, ARR3 (arrestin C), CDF10, CDKN2A,CTGF, CXCL12/SDF1, BHLHE41, BMP2, DKK1, EGFR, EPHB2, FN1, FOSL1 and 2,FOXD1, GABBR1, GAS1 and 6, GBX2, HHEX, HOXB2, IGFBP5 and 7, INHBA, JAG1,KDR, KLF1OLHX9, LHX2, LIF, MET, NEUROD1, NTF3, NTRK2, OPTN (optineurin),RCVRN, SAG (S-arrestin), SERPINF1 (PEDF), SFRP1, SOX3, TBX3, TGFB, WIF,WNT5A, and WNT5B. Markers that may be decreased relative to other CNSprogenitor cells include, without limitation, AQP4, ASLL1, CLDN11,CDKN1B, CCL2, CCNG2, CXCR4 (SDF1 receptor), DCX, DLX2 and 5, EMX2,EPHA3, 4, and 7, FABP7, FOXG1, GRIA 1, 2, and 3, HGF, IL2, KLF4, LIFR,MNX1, NGF, NKX2-2, NOTCH1, NPY, NPY2R, OLIG2, OMG, PBX1, PDGFRA, RTN1,SCGN, SOX11, TFAP2B, TNFRSF21, and WNT7A.

In alternative embodiments cell populations may be harvested fromhealthy subjects (i.e., individuals not harboring a retinal disease),from diseased subjects, and may include not only fresh retinal cellpopulations, but also frozen retinal cell populations. Sources include,without limitation, whole eyes, or retinal tissues, or other sources,obtained from embryonic, fetal, pediatric or adult tissue. The methodscan include further enrichment or purification procedures or steps forcell isolation by positive selection for other retinal progenitor cellspecific markers. The retinal progenitor cells and cell populations maybe obtained or harvested from any mammalian species or subjects, e.g.human, primate, equine, bovine, porcine, canine, feline, ferret, rabbit,rodent, e.g. mice, rats, hamster, etc.

In vertebrate embryonic development, the retina and the optic nerveoriginate as outgrowths of the developing brain, so the retina isconsidered part of the central nervous system (CNS) and is actuallybrain tissue. The retina is a layered structure with several layers ofneurons interconnected by synapses. From closest to farthest from thevitreous body, that is, from closest to the front exterior of the headtowards the interior and back of the head, the retinal layers includethe inner limiting membrane comprised of Müller cell footplates, thenerve fiber layer containing axons of the ganglion cell nuclei, theganglion cell layer, which contains nuclei of ganglion cells, the axonsof which become the optic nerve fiber, the inner plexiform layer thatcontains the synapse between the bipolar cell axons and the dendrites ofthe ganglion and amacrine cells, the inner nuclear layer, which containsthe nuclei and surrounding cell bodies (perikarya) of the bipolar cells,the outer plexiform layer, containing projections of rods and conesending in the rod spherule and cone pedicle, respectively, the outernuclear layer, which contain cell bodies of rods and cones, the externallimiting membrane, which separates the inner segment portions of thephotoreceptors from their cell nucleus, the photoreceptor layer, and theretinal pigment epithelium (RPE), which is a single layer of cuboidalcells. The neurons that are directly sensitive to light are thephotoreceptor cells, comprised mainly of two types: the rods and cones.Rods function mainly in dim light and provide black-and-white vision,while cones support daytime vision and the perception of color. A thirdtype of photoreceptor is the photosensitive ganglion cell, is importantfor reflexive responses to bright daylight.

In some embodiments, cells are harvested from a mammalian fetal retinaat a stage after which the retina is formed, but before photoreceptorouter segments are fully formed throughout the retina and before retinalvascularization has been completed or substantially completed. Thestages are typically between fetal gestational ages of about 12 weeks toabout 28 weeks in a human fetus. For non-human cells from largermammals, such as feline or porcine retinal progenitor cells, the stagesare typically between fetal gestational ages of about 3 weeks to about11 weeks. See, for example, Anand-Apte, B. and Hollyfield, J. G.“Developmental Anatomy of the Retinal and Choroidal Vasculature.” In TheRetina and Its Disorders, Besharse, J. and Bok, D., Academic Press,(2001). However, the subject matter disclosed herein also includesharvesting cells from postnatal or neonatal mammalian tissue.

In alternative embodiments retinal progenitor cells are purified fromother tissue components after or concurrent with the processing of atissue sample. In one embodiment, progenitor cells are purified fromother cells and tissue components after the tissue sample has beencultured under conditions suitable for cell growth and for a timesufficient to allow cells to adhere to the culture dish. In certainembodiments, purification of cells comprises obtaining cells thatmigrate from the tissue sample during culture and are present in theculture media or loosely adhered to a fibronectin or other substrate, ora feeder cell layer. These cells may be obtained by routine methods,such as removing and centrifuging the media to pellet cells therein, andwashing the cells remaining in the culture dish with a solution such asphosphate-buffered saline (PBS) or Hanks Balanced Salt Solution toremove those cells loosely attached as an adherent cell layer. This washsolution may then also be centrifuged to obtain cells. In someembodiments, purification of retinal progenitor cells and cellpopulations may further comprise separating cells from certain insolubletissue components, including residual tissue material, such as lipids.Cells may be separated from other tissue components by any means knownand available in the art, including, e.g., the use of density gradients,centrifugation, sorting by flow cytometry or magnetic cell separation(MACS), and filtration or combinations thereof. Examples of specificmethods of purifying cells are known and described in the art, e.g., inU.S. Pat. No. 6,777,231. In certain embodiments, negative separationmethods are employed to remove one or more particular types of cells.

In certain embodiments, tissue is processed or “dissociated”.Dissociation may be carried out by physical dissociation and/or byexposure to an enzyme preparation that facilitates the release of cellsfrom other tissue components to create a “dissociated suspension” ofcells and/or cell clusters. Examples of such enzymes include matrixmetalloproteinases, clostripain, papain, trypsin, trypsin-like, pepsin,pepsin-like, neutral protease-type and collagenases. Suitableproteolytic enzymes are described in U.S. Pat. Nos. 5,079,160;6,589,728; 5,422,261; 5,424,208; and 5,322,790. In some embodiments, theenzyme preparation includes trypsin alone or in combination with one ormore additional enzymes. Enzymatic dissociation may be carried out inconjunction with physical dissociation by, for example, mincing,pipetting, chopping, homogenizing, grinding, freeze-thawing, osmoticallyshocking, to remove unwanted cells or connective tissue and ultimatelyresulting in single cell cultures or may include cell clusters that canbe defined by size, i.e., “small”, “medium” and “large”. Cell clustersize is subjective and may vary in the practice of the subject matterdisclosed herein.

Cell culture describes a process by which cells are grown undercontrolled conditions, generally outside of their natural environment.In alternative embodiments cell populations are grown or cultured in anycell culture medium known in the art. In alternative embodiments, “Basalmedium” used in the present invention refers to any medium that cansupport cell growth. The basal medium provides standard inorganic saltssuch as zinc, iron, magnesium, calcium, and potassium, vitamins,glucose, buffer system, and key amino acids. The basal medium that canbe used in the present invention includes, but is not limited to,Minimum Essential Medium Eagle, ADC-1, LPM (Bovine Serum Albumin-free),F10 (Ham), F12 (Ham), DCCM1, DCCM2, RPMI 1640, BGJ Medium (with andwithout Fitton-Jackson Modification), Basal Medium Eagle (BME-with theaddition of Earle's salt base), Dulbecco's Modified Eagle Medium(DMEM-without serum), Yamane, IMEM-20, Glasgow Modification Eagle Medium(GMEM), Leibovitz L-15 Medium, McCoy's 5A Medium, Medium M199(M199E-with Earle's sale base), Medium M199 (M199H-with Hank's saltbase), Minimum Essential Medium Eagle (MEM-E-with Earle's salt base),Minimum Essential Medium Eagle (MEM-H-with Hank's salt base) and MinimumEssential Medium Eagle (MEM-NAA with non essential amino acids), amongnumerous others, including medium 199, CMRL 1415, CMRL 1969, CMRL 1066,NCTC 135, MB 75261, MAB 8713, DM 145, Williams' G, Neuman & Tytell,Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB 401, MCDB 411, MDBC 153, andUltraculture. Preferred media for use in culturing the retinalprogenitor cells disclosed herein are Advanced DMEM/F12 andUltraculture. A number of these media are summarized in Methods inEnzymology, Volume LVIII, “Cell Culture,” pp. 62-72.

In alternative embodiments “Conditioned medium” refers to a medium thatis altered as compared to a base or basal medium. For example, theconditioning of a medium may cause molecules, such as nutrients and/orgrowth factors, to be added to or depleted from the original levelsfound in the base medium. In some embodiments, a medium is conditionedby allowing cells of certain types to be grown or maintained in themedium under certain conditions for a certain period of time. Forexample, a medium can be conditioned by allowing retinal progenitorcells to be expanded, differentiated or maintained in a medium ofdefined composition at a defined temperature for a defined number ofhours. As will be appreciated by those of skill in the art, numerouscombinations of cells, media types, durations and environmentalconditions can be used to produce nearly an infinite array ofconditioned media.

Examples of cell culture supplements or additives include, withoutlimitation, ingredients to replace partly or wholly the role of serum insupporting cell survival or growth. In alternative embodiments, itincludes insulin, transmetalloproteins, trace elements, vitamins, orother factors. These factors are generally not included in the basalmedium but are provided by serum used generally in culturing cells. Thesupplement or additive may comprise at least one or more of thefollowing components that support cell growth: one or more insulins orreplacements thereof, one or more transmetalloproteins or replacementsthereof, one or more trace elements (e.g., selenium, iron, zinc, copper,cobalt, chromium, iodine, fluoride, manganese, molybdenum, vanadium,nickel, tin), one or more vitamins (e.g., Vitamin C, Vitamin E, VitaminA, Vitamin B-group), one or more salts (e.g., sodium salts, magnesiumsalts, calcium salts, or phosphate salts), one or more buffers (e.g.,phosphate buffered saline, HEPES buffer), one or more amino acids (e.g.,L-glutamine), one or more hormones, hormone-like compounds or growthfactors (such as, e.g., transferrin, EGF, NGF, ECGF, PDGF, FGF, IGF,LIF, interleukins, interferons, TGF, and/or VEGF, glucagon,corticosteroids, vasopressin, prostaglandins), serum albumin orreplacements thereof, one or more carbohydrates (glucose, galactose,fructose, mannose, ribose, glycolytic metabolites), one or moreantibiotics and/or antimycotics (e.g., penicillin, streptomycin,Fungizone), and one or more lipids (e.g., free and protein-bound fattyacids, triglycerides, phospholipids, cholesterol, ethanolamine). Manycommercialized serum replacement additives, such as KnockOut SerumReplacement (KOSR), N2, B27, StemPro, Insulin-Transferrin-SeleniumSupplement (ITS), and G5 are well known and are readily available tothose skilled in the art. These additives are characterized bywell-defined ingredients, so the concentrations of its components can bedetermined based on its proportion in the medium.

In alternative embodiments cultures of mammalian retinal progenitorcells can be produced in medium containing reduced serum or no serum.Examples of serum include fetal bovine serum, calf serum, newborn calfserum, goat serum, horse serum, human serum, rabbit serum, rat serum,mouse serum, among others. Under certain culture conditions, serumconcentrations can range from about 0.05% v/v to about 20% v/v. Forexample, in some differentiation processes, the serum concentration ofthe medium can be less than about 0.05% (v/v), less than about 0.1%(v/v), less than about 0.2% (v/v), less than about 0.3% (v/v), less thanabout 0.4% (v/v), less than about 0.5% (v/v), less than about 0.6%(v/v), less than about 0.7% (v/v), less than about 0.8% (v/v), less thanabout 0.9% (v/v), less than about 1% (v/v), less than about 2% (v/v),less than about 3% (v/v), less than about 4% (v/v), less than about 5%(v/v), less than about 6% (v/v), less than about 7% (v/v), less thanabout 8% (v/v), less than about 9% (v/v), less than about 10% (v/v),less than about 15% (v/v) or less than about 20% (v/v). In someembodiments, retinal progenitor cells and cell populations comprisingretinal progenitor cells are grown without serum (“serum-free”), withoutserum replacement and/or without any supplement.

In some embodiments, retinal progenitor cells or cell populationscomprising retinal progenitor cells are cultured under “xeno-free”conditions. “Xeno-free” or “xenogen-free” refers to conditions wherecells of a certain species (e.g., human cells) are grown or culturedonly in the presence of human products or supplements (e.g., human serumalbumin, human serum), but not products from other species. This isparticularly important for cells that are used for transplantation intoa human. Cells that have been exposed to a variety of undefinedanimal-derived products make them undesirable for clinical applications,because of an increased risk of graft rejection, immunoreactions, andviral or bacterial infections, prions, and yet unidentified zoonoses.Moreover, for all mammalian uses, including human use or non-humanmammalian uses (e.g., veterinary uses), cells are screened for normalkaryotype or presence of infection or contamination, e.g., bymycoplasma, gram negative bacteria (e.g., endotoxin test), fungi and thelike. Cells may also be screened for tumorigenicity or transformation toa cancerous phenotype by telomerase activity assay, hTERT geneexpression, and growth in soft agar or tumor formation in nude mice.Such assays are known in the art and well within the purview of theskilled artisan.

In alternative embodiments retinal progenitor cells or cell populationscomprising retinal progenitor cells may be cultured on feeder celllayers (e.g., embryonic or adult fibroblasts), or in the presence of anextracellular matrix scaffold or substrates such as collagen, entactin,heparin sulfate proteoglycans, fibronectin, laminin, gelatin, orMatrigel.

For example, PURECOL® collagen is known as the standard of all collagensfor purity (>99.9% collagen content), functionality, and the mostnative-like collagen available. PURECOL® collagen is approximately 97%Type I collagen with the remainder being comprised of Type III collagen,and is ideal for coating of surfaces, providing preparation of thinlayers for culturing cells, or use as a solid gel. Another example of ascaffold or substrate known in the art is CELLstart (Invitrogen).

In alternative embodiments cell culture conditions can involve growth ofcells in an incubator set at 37° C., 5% CO₂. Retinal progenitor cells orcell populations comprising retinal progenitor cells may be culturedunder normoxic or atmospheric (20%), and can be grown under conditionsthat approximate oxygen levels of a developing fetal retina duringgestation, i.e., “low” or “hypoxic” conditions, e.g., 0.5%, 1%, 1.5%,2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5% or 7% oxygen, or anyincrement in between.

Plating density refers to the number of cells per volume of culturemedium or the number of cells per cm² in adherent culture. A similarterm in this context is “confluence”, which is commonly used as ameasure of the number of the cells in a cell culture dish or a flask,and refers to the coverage of the dish or the flask by the cells. Forexample, 100 percent confluency means the dish is completely covered bythe cells, and therefore no more room left for the cells to grow;whereas 50 percent confluency means roughly half of the dish is coveredand there is still room for cells to grow.

Passaging (also known as subculture or splitting cells) involvestransferring a small number of cells into a new vessel. In alternativeembodiments cells are cultured for a longer time if they are splitregularly, as it avoids the senescence associated with prolonged highcell density. Suspension cultures are easily passaged with a smallamount of culture containing a few cells diluted in a larger volume offresh media. For adherent cultures, cells first need to be detached;this is commonly done with a mixture of an enzyme such as trypsin-EDTAor non-enzymatic solution like Cell Dissociation Buffer; however, avariety of enzyme or non-enzyme mixes or preparations are available forthis purpose. A small number of detached cells can then be used to seeda new culture.

Most primary cell cultures have limited lifespan and do not proliferateindefinitely. After a certain number of population doublings (called theHayflick limit), cells undergo the process of senescence and stopdividing, while generally retaining viability. In alternativeembodiments, the retinal progenitor cells and cell populations can becultured for no more than 10 passages, for example, are passaged one,two, three, four, five, six, seven, eight, nine, or ten passages. Inalternative embodiments, cells can be passaged more than 10 times, suchas, e.g., eleven, twelve, thirteen, fourteen, fifteen, sixteen,seventeen, eighteen, nineteen, twenty or more passages. In certainembodiments, the retinal progenitor cells and cell populations arecultured for about 10-30 passages. In alternative embodiments, retinalprogenitor cells and cell populations comprising them are preferably notimmortal, nor are they allowed to immortalize, or forced to immortalize.While the cells described herein are considered immature progenitorcells, the cell populations comprising the retinal progenitor cells maycontain cells that may be considered “multipotent” stem cells per se,wherein such cells are generally capable under certain cultureconditions of differentiating into retinal tissue or retinal cells,including retinal progenitor cells. In alternative embodiments“Multipotent” refers to immature, undifferentiated and/or unspecializedcells that have the ability to self-renew, but are limited in theability to differentiate and are essentially committed to producespecific cell types. In certain embodiments, cells and cell populationsdescribed herein comprise closely related cells, but are not necessarilyindicative of a single cell type.

In alternative embodiments retinal progenitor cells are geneticallymodified to express one or more heterologous or exogenous nucleic acidsequences of interest. A nucleic acid sequence can be “exogenous,” whichmeans that it is foreign to the cell into which the vector is beingintroduced or that the sequence is homologous to a sequence in the cellbut in a position within the host cell nucleic acid in which thesequence is ordinarily not found. Nucleic acid sequences includeplasmids, amplicons, cDNA, mRNA, antisense RNA, siRNA, but are notlimited to these examples. The term “gene” refers to a functionalprotein, polypeptide, or peptide-encoding nucleic acid unit. As will beunderstood by those in the art, this functional term includes genomicsequences, cDNA sequences, and smaller engineered gene segments thatexpress, or may be adapted to express, proteins, polypeptides, domains,peptides, fusion proteins, and mutants.

Any methodology known in the art can be used for genetically alteringthe tissue. One exemplary method is to insert a gene into the cells ofthe tissue with a recombinant viral vector. Any one of a number ofdifferent vectors can be used, such as viral vectors, plasmid vectors,linear DNA, etc., as known in the art, to introduce an exogenous nucleicacid fragment encoding for a therapeutic agent into target cells and/ortissue. These vectors can be inserted, for example, using any ofinfection, transduction, transfection, calcium-phosphate mediatedtransfection, DEAE-dextran mediated transfection, electroporation,liposome-mediated transfection, biolistic gene delivery, liposomal genedelivery using fusogenic and anionic liposomes (which are an alternativeto the use of cationic liposomes), direct injection, receptor-mediateduptake, magnetoporation, ultrasound and others as known in the art.

In alternative embodiments a “vector” is used to refer to a carriernucleic acid molecule into which a nucleic acid sequence can be insertedfor introduction into a cell where it can be replicated. Vectors includeplasmids, cosmids, viruses (bacteriophage, animal viruses, and plantviruses), and artificial chromosomes (e.g., YACs). One of skill in theart would be well equipped to construct a vector through standardrecombinant techniques, which are described in Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y.;Ausubel et al. (1987) Current Protocols in Molecular Biology, GreenePubl. Assoc. & Wiley-Intersciences. In addition to encoding a modifiedpolypeptide, a vector may encode non-modified polypeptide sequences suchas a tag or targeting molecule. Useful vectors encoding such fusionproteins include pIN vectors, vectors encoding a stretch of histidines,and pGEX vectors, for use in generating glutathione S-transferase (GST)soluble fusion proteins for later purification and separation orcleavage.

In alternative embodiments vectors of the present invention are designedprimarily to introduce into cells a heterologous nucleic acid molecule,such as a gene that is “operably linked” or under the control of one ormore control sequences. A “promoter” refers to one or moretranscriptional control modules that are clustered around the initiationsite for RNA polymerase II and other transcriptional activator proteins.Any promoter/enhancer combination (as per the Eukaryotic Promoter DataBase EPDB) could also be used to drive expression of a nucleic acidmolecule of interest (i.e., constitutive, inducible, repressible, tissuespecific). Also, the vectors may contain a selectable marker tofacilitate their manipulation in vitro or ex vivo. Vectors may alsocontain a polyadenylation signal, which may be obtained from the humangrowth hormone (hGH) gene, the bovine growth hormone (BGH) gene, orSV40. In addition, vectors may also contain internal ribosome bindingsites (IRES) elements are used to create multigene, or polycistronic,messages. IRES elements are able to bypass the ribosome scanning modelof 5-methylatd cap-dependent translation and begin translation atinternal sites (Pelletier, J. and Sonenberg, N. (1988) Nature 334(6180):

320-325). IRES elements can be linked to heterologous open readingframes. Multiple open reading frames can be transcribed together, eachseparated by an IRES, creating polycistronic messages. By virtue of theIRES element, each open reading frame is accessible to ribosomes forefficient translation. Multiple genes can be efficiently expressed usinga single promoter/enhancer to transcribe a single message.

In alternative embodiments, a vector is a viral vector. Viral vectorsknown in the art include, without limitation, adenoviral vectors,retroviral vectors, vaccinia viral vectors, adeno-associated viralvectors, polyoma viral vectors, alphaviral vectors, rhabdoviral vectors,lentiviral vectors, Epstein-Barr viral vectors, picornaviral vectors, orherpesviral vectors.

In other embodiments, a nucleic acid sequence may be entrapped in aliposome or lipid formulation. Liposomes are vesicular structurescharacterized by a phospholipid bilayer membrane and an inner aqueousmedium. Multilamellar liposomes have multiple lipid layers separated byaqueous medium. They form spontaneously when phospholipids are suspendedin an excess of aqueous solution. The lipid components undergoself-rearrangement before the formation of closed structures and entrapwater and dissolved solutes between the lipid bilayers (Ghosh, P. C. andBachhawat, B. K. (1991) Targeted Diagn. Ther. 4: 87-103). One example ofa commercially available liposomes or lipid formulations isLipofectamine (Invitrogen). Others include FuGENE (Promega), PromoFectin(PromoKine), Affectene (Qiagen), Polyfect (Qiagen), Superfect (Qiagen),and TransMessenger (Qiagen).

The subject matter disclosed herein also provides methods of treating aretinal disease or condition in a subject in need thereof byadministering to the subject a composition comprising a cell populationcomprising mammalian retinal progenitor cells into a subject's vitreouscavity or subretinal space, and optionally measuring changes orimprovements in vision in the subject.

In alternative embodiments the term “treating” in its variousgrammatical forms in relation to the present invention refers to curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of a disease state, disease progression, diseasecausative agent or other abnormal condition. For example, treatment mayinvolve alleviating a symptom (i.e., not necessary all symptoms) of adisease or attenuating the progression of a disease. In alternativeembodiments the term “preventing” means that the effects of a diseasestate or disease causative agent has been obviated due to administrationof an agent, such as those disclosed herein. A similar term in thiscontext is “prophylaxis”.

“Patient” or “subject” are used interchangeably herein and refer to therecipient of treatment. Mammalian and non-mammalian subjects areincluded. In some embodiment, the subject is a mammal, such as a human,non-human primate, canine, murine, feline, bovine, ovine, porcine, orcaprine. In some embodiments, the subject is a human.

Cell populations and related compositions described herein may beprovided to a subject or patient by a variety of different means. Incertain embodiments, they are provided locally, e.g., to a site ofactual or potential injury or disease. In some embodiment, they areprovided using a syringe or needle to inject the compositions at a siteof possible or actual injury or disease. In other embodiments, they areprovided systemically, i.e., administered to the bloodstreamintravenously or intra-arterially. The particular route ofadministration will depend, in large part, upon the location and natureof the disease or injury being treated or prevented. Accordingly, thesubject matter described herein includes providing a cell population orcomposition of the invention via any known and available method orroute, including but not limited to oral, parenteral, intravenous,intra-arterial, intranasal, and intramuscular administration. Thedetermination of suitable dosages and treatment regimens may be readilyaccomplished based upon information generally known in the art andobtained by a physician. Treatment may comprise a single treatment ormultiple treatments. In particular, for preventative purposes, it iscontemplated in certain embodiments that purified cell populations ofthe invention are administered following a stress that might potentiallycause retinal injury. In other embodiments, the cell populations andcompositions may be locally administered as a single injection to thevitreous cavity or subretinal space of the subject. Alternatively, thecompositions or cell populations may be administered two times, threetimes, four times, or any number of times in the practice of the methodsprovided herein.

In alternative embodiments methods of the invention comprise isolationand characterization of mammalian retinal progenitor cells andcompositions comprising such cells that are harvested from donor tissue,grown in culture, and formulated for administration to a subject orpatient. In some embodiments, the methods comprise administering thecompositions under topical anesthesia directly to the vitreous cavity,without need for systemic immune suppression of the subject. Inalternative embodiments, tissue typing of graft and matching to patientor subject is not required, but may be performed if desired. Tissuetyping and matching techniques are well known to those skilled in theart.

The retinal progenitor cells and cell populations used to practice thisinvention may be formulated as a composition for administration by anyor a variety of means including orally, parenterally, by inhalationspray, nasally, topically, intrathecally, intrathecally,intracerebrally, epidurally, intracranially or rectally. Compositionsand formulations disclosed herein can comprise pharmaceutically orveterinarily acceptable liquids, carriers, adjuvants and vehicles andcan be in the form of liquids, tablets, capsules, implants, aerosols,gels, liposomes, nanoparticles and the like.

In some embodiments, the retinal progenitor cells and cell populationsmay be administered to a subject in the form of pharmaceutical orveterinary compositions. The phrase “pharmaceutically acceptable” refersto molecular entities and compositions that do not produce adverse,allergic, or other untoward reactions when administered to an animal ora human. As used herein, “pharmaceutically acceptable carrier” includesany and all aqueous and nonaqueous carriers which includes water,ethanol, polyols (such as glycerol, propylene glycol, polyethyleneglycol, and the like), and suitable mixtures thereof, vegetable oils,such as olive oil, and injectable organic esters, such as ethyl oleate.Proper fluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants. Thesecompositions may also contain adjuvants such as preservatives, wettingagents, emulsifying agents and dispersing agents. Prevention of thepresence of microorganisms may be ensured both by sterilizationprocedures and by the inclusion of various antibacterial and antifungalagents, for example, paraben, chlorobutanol, sorbic acid, and the like.It may also be desirable to include isotonic agents, such as sugars,sodium chloride, and the like into the compositions. In addition,prolonged absorption of the injectable pharmaceutical form may bebrought about by the inclusion of agents which delay absorption such asaluminum monostearate and gelatin. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Theuse of such media and agents for pharmaceutically active substances iswell known in the art.

In certain embodiments, an effective amount of the retinal progenitorcells or cell populations must be administered to the subject. An“effective amount” or “therapeutically effective amount” refers to theamount of the composition that produces a desired effect. An effectiveamount will depend, for example, in part, upon the molecule or agentdelivered (here the retinal progenitor cells or cell populations), theindication for which the therapeutic agent is being used, the route ofadministration, and the size (body weight, body surface or organ size)and condition (the age and general health) of the subject or patient.Accordingly, the clinician or physician may titer the dosage and modifythe route of administration to obtain the optimal therapeutic effect. Aneffective amount of a particular agent for a specific purpose can bedetermined using methods well known to those in the art. For anycomposition defined herein, the effective amount can be estimatedinitially either in cell culture assays or in animal models such asmice, rats, rabbits, dogs, pigs, or monkeys. An animal model may also beused to determine the appropriate concentration range and route ofadministration. Such information can then be used to determine usefuldoses and routes for administration in humans.

Examples of effective amounts of the compositions described hereininclude cell suspensions at a volume of 5 μl, 10 μl, 15 μl, 20 μl, 25μl, 50 μl, 100 μl, 150 μl, 200 μl, 250 μl, 300 μl, 350 μl, 400 μl, 450μl, 500 μl or any increment in between up to 5000 μl (5 ml). The lowerand upper volume limits are limited by the delivery system and/ormethod. See, e.g., Kayikcuiglu, O. R. et al, (2006) Retina 26(9):1089-90. For example, the upper volume limit when administered withoutvitrectomy is approximately 200 μl due to increased intraocularpressure. The upper volume limit when administered with vitrectomy intothe vitreous cavity is limited by the volume of the vitreous cavity andcan comprise up to 5 ml or more. The upper limit for subretinalinjection may be up to 200 μl due to retinal detachment. In alternativeembodiments, these volumes include anywhere between 1000 to 10 millioncells per dose, or 1000 to 2000 cells per dose, 2000 to 3000 cells perdose, 3000 to 4000 cells per dose, 400 to 5000 cells per dose, 5000 to6000 cells per dose, 6000 to 7000 cells per dose, 7000 to 8000 cells perdose, 8000 to 9000 cells per dose, 9000 to 10,000 cells per dose, 10,000to 15,000 cells per dose, 15,000 to 20,000 cells per dose, 20,000 to25,000 cells per dose, 25,000 to 30,000 cells per dose, 30,000 to 35,000cells per dose, 35,000 to 40,000 cells per dose, 40,000 to 45,000 cellsper dose, 45,000 to 50,000 cells per dose, 50,000 to 55,000 cells perdose, 55,000 to 60,000 cells per dose, 60,000 to 65,000 cells per dose,65,000 to 70,000 cells per dose, 70,000 to 75,000 cells per dose, 75,000to 80,000 cells per dose, 80,000 to 85,000 cells per dose, 85,000 to90,000 cells per dose, 90,000 to 95,000 cells per dose, 95,000 to100,000 cells per dose, 100,000 to 125,000 cells per dose, 125,000 to150,000 cells per dose, 150,000 to 200,000 cells per dose, 200,000 to250,000 cells per dose, 250,000 to 300,000 cells per dose, 300,000 to350,000 cells per dose, 350,000 to 400,000 cells per dose, 400,000 to450,000 cells per dose, 450,000 to 500,000 cells per dose, 500,000 to550,000 cells per dose, 550,000 to 600,000 cells per dose, 600,000 to650,000 cells per dose, 650,000 to 700,000 cells per dose, 700,000 to750,000 cells per dose, 750,000 to 800,000 cells per dose, 800,000 to850,000 cells per dose, 850,000 to 900,000 cells per dose, 900,000 to950,000 cells per dose, 950,000 to 1,000,000 cells per dose or in anyincrement in between 1000 cells and up to 10 million cells per dose.Dosages may, of course, vary according to frequency and duration ofadministration. In alternative embodiments the dosage of cells in thecompositions described herein contains a high number of cells in a smallvolume, such as, for example, 0.5 million cells per 100 μl. Cell numbersmay be counted by any method known in the art, such as by hemacytometer,spectrophotometry, Coulter counter, flow cytometry, etc. Dosing may beadministered once or may be administered over the course of severaltreatments.

In alternative embodiments compositions of the invention can beformulated for parenteral administration into the eye (particularly intothe vitreous cavity or subretinal space), a vitreous cavity or asubretinal space, retina, brain, nerve or CNS by transscleral delivery,or by any method or protocol known in the art, e.g., including atransscleral delivery as described in U.S. Pat. No. 7,585,517; asustained release delivery device for delivery to the interior of apatient's eye as described in U.S. Pat. No. 7,883,717; a device forinsertion in the vitreous region of the eye as described in U.S. Pat.Nos. 5,378,475 or 5,466,233; or by use of a hypodermic syringe or angledinsertion pathway, e.g., as described in U.S. Patent ApplicationPublication Nos. 20110112470 or 20100256597 (describing a microneedlefor targeted administration to a patient's eye); or via a hydrophilicpolymer hydrogel with dimensions to pass through a puncta lacrimalie.g., as described in U.S. Patent Application Publication No.20100209478; or a device that provides access to the sub-retinal spacein a human eye e.g., as described in U.S. Patent Application PublicationNo. 20100191176. Anterior chamber paracentesis also can be performed asdetermined by one of skill in the art. In alternative embodiments,methods do not require suturing of globe during and/or after aprocedure, particularly for intravitreal placement. However, this may benecessary for methods utilizing a vitrectomy procedure, for example,when placing cells in the subretinal space.

The compositions disclosed herein may also be formulated forintrathecal, intracerebral epidural, subcutaneous, intravenous,intramuscular and/or intraarterial administration; e.g., as described inU.S. Patent Application Publication No. 200500480021; by injectionroutes but also including a variety of infusion techniques.Administration may be carried out through the use of catheters or pumps,e.g., an intrathecal pump, or an implantable medical device. Inalternative embodiments methods of the invention also may involveadministration or transplantation of implants and artificial organs,bioreactor systems, cell culture systems, plates, dishes, tubes, bottlesand flasks and the like, comprising the retinal progenitor cells, cellpopulations, or compositions disclosed herein, such as those describedin U.S. Pat. Nos. 7,388,042; 7,381,418; 7,379,765; 7,361,332; 7,351,423;6,886,568; 5,270,192; and U.S. Patent Application Publication Nos.20040127987; 20080119909; 20080118549; 20080020015; 20070254005;20070059335; 20060128015.

In alternative embodiments methods provided herein may be used fortreating, ameliorating or preventing a retinal disease or condition,such as, without limitation, retinitis pigmentosa (RP), Leber'scongenital amaurosis (LCA), Stargardt disease, Usher's syndrome,choroideremia, a rod-cone or cone-rod dystrophy, a ciliopathy, amitochondrial disorder, progressive retinal atrophy, a degenerativeretinal disease, age related macular degeneration (AMD), wet AMID, dryAMID, geographic atrophy, a familial or acquired maculopathy, a retinalphotoreceptor disease, a retinal pigment epithelial-based disease,diabetic retinopathy, cystoid macular edema, uveitis, retinaldetachment, traumatic retinal injury, iatrogenic retinal injury, macularholes, macular telangiectasia, a ganglion cell disease, an optic nervecell disease, glaucoma, optic neuropathy, ischemic retinal disease,retinopathy of prematurity, retinal vascular occlusion, familialmacroaneurysm, a retinal vascular disease, an ocular vascular diseases,a vascular disease, or ischemic optic neuropathy; for improving aphotopic (day light) vision; or for improving correcting visual acuity,or improving macular function, or improving a visual field, or improvingscotopic (night) vision.

In alternative embodiments treatment methods of the invention utilizetopical anesthesia, however any local, regional or general anesthesiamay be used during administration. Examples of local anestheticssuitable for use in the methods disclosed herein include, withoutlimitation, mepricaine, proparacaine, prilocaine, ropivacaine,benzocaine, bupivacaine, butamben picrate, chlorprocaine, cocaine,dibucaine, dimethisoquin, dyclonine, etidocaine, hexylcaine, ketamine,lidocaine, mepivacaine, pramoxine, procaine, tetracaine, salicylates andderivatives, esters, salts and mixtures thereof.

The compositions comprising retinal progenitor cells or cell populationsmay optionally be co-administered with one or more drugs. Examples ofdrugs may include anti-angiogenesis agents such as angiostatin,anecortave acetate, thrombospondin, VEGF receptor tyrosine kinaseinhibitors and anti-vascular endothelial growth factor (anti-VEGF) drugssuch as ranibizumab and bevacizumab, pegaptanib, sunitinib and sorafeniband any of a variety of known small-molecule and transcriptioninhibitors having anti-angiogenesis effect; classes of known ophthalmicdrugs, including: glaucoma agents, such as adrenergic antagonists,including for example, beta-blocker agents such as acetbutolol,atenolol, bisoprolol, carvedilol, asmolol, labetalol, nadolol,penbutolol, pindolol, propranolol, metipranolol, betaxolol, carteolol,levobetaxolol, levobunolol and timolol; adrenergic agonists orsympathomimetic agents such as epinephrine, dipivefrin, clonidine,aparclonidine, and brimonidine; parasympathomimetics or cholingericagonists such as pilocarpine, carbachol, phospholine iodine, andphysostigmine, salicylate, acetylcholine chloride, eserine, diisopropylfluorophosphate, demecarium bromide); muscarinics; carbonic anhydraseinhibitor agents, including topical and/or systemic agents, for exampleacetozolamide, brinzolamide, dorzolamide and methazolamide,ethoxzolamide, diamox, and dichlorphenamide; mydriatic-cycloplegicagents such as atropine, cyclopentolate, succinylcholine, homatropine,phenylephrine, scopolamine and tropicamide; prostaglandins such asprostaglandin F2 alpha, antiprostaglandins, prostaglandin precursors, orprostaglandin analog agents such as bimatoprost, latanoprost, travoprostand unoprostone.

Other examples of drugs may also include anti-inflammatory agentsincluding for example glucocorticoids and corticosteroids such asbetamethasone, cortisone, dexamethasone, dexamethasone 21-phosphate,methylprednisolone, prednisolone 21-phosphate, prednisolone acetate,prednisolone, fluroometholone, loteprednol, medrysone, fluocinoloneacetonide, triamcinolone acetonide, triamcinolone, triamcinoloneacetonide, beclomethasone, budesonide, flunisolide, fluorometholone,fluticasone, fludrocortisone, hydrocortisone, hydrocortisone acetate,loteprednol, rimexolone and non-steroidal anti-inflammatory agentsincluding, for example, aspirin, diclofenac, flurbiprofen, ibuprofen,bromfenac, nepafenac, and ketorolac, salicylate, indomethacin, naxopren,piroxicam and nabumetone diflunisal, etodolac, fenoprofen, flurbiprofen,indomethacine, ketoprofen, meclofenamate, mefenamic acid, meloxicam,nabumetone, oxaprozin, piroxicam, salsalate, sulindac and tolmetin;COX-2 inhibitors like celecoxib, rofecoxib and Valdecoxib;anti-infective or antimicrobial agents such as antibiotics including,for example, tetracycline, chlortetracycline, bacitracin, neomycin,polymyxin, gramicidin, cephalexin, oxytetracycline, chloramphenicol,rifampicin, ciprofloxacin, tobramycin, gentamycin, erythromycin,penicillin, sulfonamides, sulfadiazine, sulfacetamide, sulfamethizole,sulfisoxazole, nitrofurazone, sodium propionate, aminoglycosides such asgentamicin, tobramycin, amikacin and streptomycin; fluoroquinolones suchas ciprofloxacin, gatifloxacin, levofloxacin, moxifloxacin, norfloxacin,ofloxacin; bacitracin, erythromycin, fusidic acid, neomycin, polymyxinB, gramicidin, trimethoprim and sulfacetamide; antifungals such asamphotericin B, caspofungin, clotrimazole, fluconazole, itraconazole,ketoconazole, voriconazole, terbinafine, nystatin and miconazole;anti-malarial agents such as chloroquine, atovaquone, mefloquine,primaquine, quinidine and quinine; anti-mycobacterium agents such asethambutol, isoniazid, pyrazinamide, rifampin and rifabutin;anti-parasitic agents such as albendazole, mebendazole, thiobendazole,metronidazole, pyrantel, atovaquone, iodoquinaol, ivermectin, paromycin,praziquantel, and trimatrexate;

Other examples of drugs may also include antiviral agents such asidoxuridine trifluorothymidine, acyclovir, cidofovir, famciclovir,gancyclovir, valacyclovir, valganciclovir, vidarabine, trifluridine andfoscarnet; protease inhibitors such as ritonavir, saquinavir, lopinavir,indinavir, atazanavir, amprenavir and nelfinavir;

nucleotide/nucleoside/non-nucleoside reverse transcriptase inhibitorssuch as abacavir, ddl, 3TC, d4T, ddC, tenofovir and emtricitabine,delavirdine, efavirenz and nevirapine; other anti-viral agents such asinterferons, ribavirin and trifluridiene; anti-bacterial agents,including cabapenems like ertapenem, imipenem and meropenem;cephalosporins such as cefadroxil, cefazolin, cefdinir, cefditoren,cephalexin, cefaclor, cefepime, cefoperazone, cefotaxime, cefotetan,cefoxitin, cefpodoxime, cefprozil, ceftaxidime, ceftibuten, ceftizoxime,ceftriaxone, cefuroxime and loracarbef; other macrolides and ketolidessuch as azithromycin, clarithromycin, dirithromycin and telithromycin;penicillins (with and without clavulanate) including amoxicillin,ampicillin, pivampicillin, dicloxacillin, nafcillin, oxacillin,piperacillin, and ticarcillin; tetracyclines such as doxycycline,minocycline and tetracycline; other anti-bacterials such as aztreonam,chloramphenicol, clindamycin, linezolid, nitrofurantoin and vancomycin;.

Other examples of drugs may also include immune-modulating agents suchas antiallergenics, including, for example, sodium chromoglycate,antazoline, methapyriline, chlorpheniramine, cetrizine, pyrilamine,prophenpyridamine; aldesleukin, adalimumab, azathioprine, basiliximab,daclizumab, etanercept, hydroxychloroquine, infliximab, leflunomide,methotrexate, mycophenolate mofetil, and sulfasalazine; anti-histamineagents such as azelastine, emedastine, loratadine, desloratadine,cetirizine, diphenhydramine, chlorpheniramine, dexchlorpheniramine,clemastine, cyproheptadine, fexofenadine, hydroxyzine, promethazine andlevocabastine; immunological drugs (such as vaccines and immunestimulants); MAST cell stabilizer agents such as cromolyn sodium,ketotifen, lodoxamide, nedocrimil, olopatadine and pemirolastciliarybody ablative agents, such as gentimicin and cidofovir; and otherophthalmic agents such as verteporfin, proparacaine, tetracaine,cyclosporine and pilocarpine; inhibitors of cell-surface glycoproteinreceptors; decongestants such as phenylephrine, naphazoline,tetrahydrazoline; lipids or hypotensive lipids; dopaminergic agonistsand/or antagonists such as quinpirole, fenoldopam, and ibopamine;vasospasm inhibitors; vasodilators; antihypertensive agents; angiotensinconverting enzyme (ACE) inhibitors; angiotensin-1 receptor antagonistssuch as olmesartan; microtubule inhibitors; molecular motor (dyneinand/or kinesin) inhibitors; actin cytoskeleton regulatory agents such ascyctchalasin, latrunculin, swinholide A, ethacrynic acid, H-7, andRho-kinase (ROCK) inhibitors; remodeling inhibitors; modulators of theextracellular matrix such as tert-butylhydro-quinolone and AL-3037A;adenosine receptor agonists and/or antagonists such asN-6-cylclophexyladenosine and (R)-phenylisopropyladenosine; serotoninagonists; hormonal agents such as estrogens, estradiol, progestationalhormones, progesterone, insulin, calcitonin, parathyroid hormone,peptide and vasopressin hypothalamus releasing factor; growth factorantagonists or growth factors, including, for example, epidermal growthfactor, fibroblast growth factor, platelet derived growth factor,transforming growth factor beta, somatotrapin, fibronectin, connectivetissue growth factor, bone morphogenic proteins (BMPs); cytokines suchas interleukins, CD44, cochlin, and serum amyloids, such as serumamyloid A.

Other therapeutic agents may include neuroprotective agents such aslubezole, nimodipine and related compounds, and including blood flowenhancers, sodium channels blockers, glutamate inhibitors such asmemantine, neurotrophic factors, nitric oxide synthase inhibitors; freeradical scavengers or anti-oxidants; chelating compounds;apoptosis-related protease inhibitors; compounds that reduce new proteinsynthesis; radiotherapeutic agents; photodynamic therapy agents; genetherapy agents; genetic modulators; and dry eye medications such ascyclosporine A, delmulcents, and sodium hyaluronate; alpha blockeragents such as doxazosin, prazosin and terazosin; calcium-channelblockers such as amlodipine, bepridil, diltiazem, felodipine,isradipine, nicardipine, nifedipine, nisoldipine and verapamil; otheranti-hypertensive agents such as clonidine, diazoxide, fenoldopan,hydralazine, minoxidil, nitroprusside, phenoxybenzamine, epoprostenol,tolazoline, treprostinil and nitrate-based agents; anti-coagulantagents, including heparins and heparinoids such as heparin, dalteparin,enoxaparin, tinzaparin and fondaparinux; other anti-coagulant agentssuch as hirudin, aprotinin, argatroban, bivalirudin, desirudin,lepirudin, warfarin and ximelagatran; anti-platelet agents such asabciximab, clopidogrel, dipyridamole, optifibatide, ticlopidine andtirofiban.

Other therapeutic agents may include prostaglandin PDE-5 inhibitors andother prostaglandin agents such as alprostadil, carboprost, sildenafil,tadalafil and vardenafil; thrombin inhibitors; antithrombogenic agents;anti-platelet aggregating agents; thrombolytic agents and/orfibrinolytic agents such as alteplase, anistreplase, reteplase,streptokinase, tenecteplase and urokinase; anti-proliferative agentssuch as sirolimus, tacrolimus, everolimus, zotarolimus, paclitaxel andmycophenolic acid; hormonal-related agents including levothyroxine,fluoxymestrone, methyltestosterone, nandrolone, oxandrolone,testosterone, estradiol, estrone, estropipate, clomiphene,gonadotropins, hydroxyprogesterone, levonorgestrel, medroxyprogesterone,megestrol, mifepristone, norethindrone, oxytocin, progesterone,raloxifene and tamoxifen; anti-neoplastic agents, including alkylatingagents such as carmustine lomustine, melphalan, cisplatin,fluorouracil3, and procarbazine antibiotic-like agents such asbleomycin, daunorubicin, doxorubicin, idarubicin, mitomycin andplicamycin; anti proliferative agents (such as 1,3-cis retinoic acid,5-fluorouracil, taxol, rapamycin, mitomycin C and cisplatin);antimetabolite agents such as cytarabine, fludarabine, hydroxyurea,mercaptopurine and 5-flurouracil (5-FU); immune modulating agents suchas aldesleukin, imatinib, rituximab and tositumomab; mitotic inhibitorsdocetaxel, etoposide, vinblastine and vincristine; radioactive agentssuch as strontium-89; and other anti-neoplastic agents such asirinotecan, topotecan and mitotane.

The uses and methods described herein can rapidly and sustainablyrestore and/or preserve clinically significant degrees of visualfunction in a retina in mammalian subjects, including but not limitedto, improved photopic (day light) vision, increased best-correctedvisual acuity, improved macular function, preserving or regainingcentral fixation, improved visual field, improvements in scotopic(night) vision, increased or improved sensitivity to sound, andimprovements in visual acuity in a contralateral eye. Other changes mayinclude various systemic benefits, e.g., changes in appearance due tosomatic improvements, which could be related to effect of light oncircadian rhythms, pituitary function, release of hormones, vasculartone; an improved sense of visual capabilities; improved ambulatoryindependence; improved sense of well-being; and/or improved activitiesof daily living. Such changes in vision can be measured by methods knownin the art. Visual benefits may occur within first week post-treatment,but may also occur as incremental benefits over longer periods of time.Retinal cell replacement and/or donor cell migration into the retina,retinal circuitry, or outer nuclear layer or macula may occur, but isnot required for clinical efficacy; donor cell migration into retina ispossible, but not required for clinical efficacy.

Measuring changes in vision, including improvements in vision resultingfrom treatment with the retinal progenitor cell compositions disclosedherein can be achieved using standard ophthalmic examination techniques,including but not limited to, fundus examination, best corrected visualacuity (BCVA), IOP, slit lamp examination, fluorescein angiography (FA),Optical Coherence Tomography (OCT), stereo-fundus photography,electroretinography (ERG), cone flicker electroretinography, perimetry(visual field), microperimetry, dark adaptation, maze negotiating skill,optokinetic/optomotor responses, pupillary responses, visual evokedpotentials (VIP), and adaptive optics scanning laser ophthalmoscopy(AOSLO).

Kits and Instructions

The invention provides kits comprising compositions (e.g., aheterogeneous mixture of fetal neural retinal cells) and methods of theinvention (e.g., treating a retinal disease or condition, or making aheterogeneous mixture of fetal neural retinal cell), includinginstructions for use thereof In alternative embodiments, the inventionprovides kits comprising a composition, product of manufacture, ormixture or culture of cells (e.g., heterogeneous mixture of fetal neuralretinal cells) of the invention; wherein optionally the kit furthercomprises instructions for practicing a method of the invention.

The invention provides kits comprising a cell population comprising themammalian retinal progenitor cells described herein, whether provided ascells in culture, fresh or frozen, or formulated as a composition foradministration into a subject. The kit may further comprise instructionsfor practicing a method of the invention. Such kits may additionalcomprise an agent that binds one or more marker of retinal progenitorcells described herein (e.g., an antibody or oligonucleotide primer),and basal or conditioned medium. For example, a kit may include: a firstcontainer comprising an antibody specific for one or more markers,wherein said antibody is adapted for isolation or detection, e.g., bybeing conjugated to a fluorescent marker or magnetic bead; and a secondcontainer comprising basal or conditioned medium. In various relatedembodiments, the kits may further comprise one or more additionalreagents useful in the preparation of a cell population of the presentinvention, such as cell culture medium, extracellular matrix-coated cellculture dishes, and enzymes suitable for tissue processing. The kit mayalso include instructions regarding its use to purify and expand theretinal progenitor cells or cell populations obtained from a tissuesample. In other embodiments, the kits may further comprise a means forobtaining a tissue sample from a patient or donor, and/or a container tohold the tissue sample obtained.

Veterinary Applications

In alternative embodiments, compositions and methods of the inventioncan be used for veterinary applications; e.g., this inventiondemonstrates the first successful growing of feline RPCs, and the firsttherapeutic application to the retina in a dystrophic cats and otheranimals, e.g. any mammalian pet, common domesticated and rare wildmammalian species, zoo animals, farm animals, sport (e.g., racing dogsor horses) animals, and the like.

There are a number of domesticated animals that harbor genes causingblindness as a result of extensive inbreeding. These included cats,dogs, and horses, and probably other species. There are retinal diseasesand injuries that occur in wild and domestic animals that will benefitfrom treatment using compositions and methods of the invention.

Products of Manufacture, Implants and Artificial Organs

The invention also provides implants and artificial organs, bioreactorsystems, cell culture systems, plates, dishes, tubes, bottles and flasksand the like comprising one more formulations or pharmaceuticals of theinvention comprising a heterogeneous mixture of fetal neural retinalcells. In alternative embodiments the invention provides a bioreactor,implant, stent, artificial organ or similar devices comprising aheterogeneous mixture of fetal neural retinal cells; for example,implants analogous to or as described in U.S. Pat. Nos. 7,388,042;7,381,418; 7,379,765; 7,361,332; 7,351,423; 6,886,568; 5,270,192; andU.S. Pat. App. Pub. Nos. 20040127987; 20080119909 (describing auricularimplants); 20080118549 (describing ocular implants); 20080020015(describing a bioactive wound dressing); 20070254005 (describing heartvalve bio-prostheses, vascular grafts, meniscus implants); 20070059335;20060128015 (describing liver implants).

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES Example 1 Isolation and Culture of Retinal Progenitor Cells

Whole fetal eyes of approximately 16 to 19 weeks gestational age (GA)fetuses were obtained from donors and placed in a 15 ml tube containingRPMI-1640 medium with L-glutamine (BioWhittaker). The eyes were shippedon ice for a period of 4.5 hours to approximately 21.5 hours. At thesame time, blood samples from the donor were drawn and sent for testingfor exposure to adventitious agents. On arrival, each eyeball wasexamined to ensure that the corneas were clear and of normal shape, thenplaced under a laminar flow hood under sterile conditions. Whole fetaleyes were rinsed three times in 40 ml cold phosphate-buffered saline(PBS) containing antibiotics in separate 50 ml tubes. The optic nerveand remaining mesenchymal tissue were then removed by dissection. Thisapproach was taken to avoid possible contamination of retinal isolateswith unwanted cells of non-retinal origin. After dissection, the eyeswere rinsed one more time in cold PBS containing antibiotics.

Under a binocular dissection microscope, a hole was punctured in eacheye at the surgical limbus using a 1 ml “TB”-type syringe mounted with25-5/8 gauge needle. The eyeball was then opened circumferentially bycutting with fine scissors along the limbus. The anterior structures(cornea, lens) and any remaining vitreous body were removed from the eyecup. The retina was then freed by carefully teasing away from theretinal pigment epithelium (RPE), and transferred into a small petridish containing approximately 2 ml cold DMEM/F12 medium. The retinaltissue was manually broken into small pieces by gentle triturationthrough a 1 ml tip in the Petri-dish. The retinal chunks weretransferred in suspension into a 15 ml cold conical bottom tube, and anyremaining tissue was collected by rinsing the petri dish 2-3 times in 1ml cold DMEM/F12 and adding it to the 15 ml tube. The tissue was spundown by centrifugation at 1000 rpm (179×g) for 5 minutes and thesupernatant was discarded.

The tissues were then subjected to enzymatic digestion by incubation in0.8 ml undiluted TrypLE Express (Invitrogen) for 40 seconds at roomtemperature. The trypsinized tissues were then drawn up and down througha 1 ml pipette tip. The trypsin was neutralized by subsequently adding10 ml of cold fresh serum-free cell culture medium and the mixturecollected by centrifugation at 1000 rpm (179×g) for 4 minutes. Thesupernatant was removed and the pellet resuspended in cold fresh cellculture medium, then cell viability and cell number were determined byTrypan blue (Invitrogen) dye exclusion, and counted using Countess(Invitrogen) or manually. Approximately 10×10⁶ cell clusters wereobtained, wherein approximately 80% were small/medium clusters,approximately 9 to 18% were single cells, and approximately 1 to 2% werebig clusters. This technique resulted in around 92% cell viability.

Cells were then seeded into two T75 culture flasks previously coatedwith human (xeno-free) fibronectin. Human plasma fibronectin(Invitrogen) was used in some experiments. In other experiments,ornithine, polylysine, laminin or Matrigel were used. The cells werethen incubated at 37° C. under 5% CO₂ and atmospheric oxygen oralternatively, in 3% O₂ using a LowOx incubator. During culture, carewas taken to further dissociate the clusters by trypsinization and/ortrituration to prevent premature differentiation as the cells aresubsequently passaged. Every one or two days, 90% of the cell culturemedium was changed and cells were passaged at 60-80% confluence,optionally at 40-90% confluence, using TrypLE Express for 5 to 6 minutesat 37° C. Trypsinization was stopped by adding 10 ml of cold medium orcold PBS. Cell viability was determined by Trypan blue staining, andcell number was counted. Dissociated cells were subsequently seeded intonew fibronectin-coated flasks or plates at a density of 1 to6.7×10⁴/cm².

Cells were prepared for freezing by first harvesting them with TrypLEExpress. The cells were collected by centrifugation at 1000 rpm (179×g)for 5 minutes. The supernatant was removed and the cell pelletresuspended in fresh medium or cold PBS. Cell viability and cell numberwere determined. The cells were subsequently spun down again at 1000 rpmfor 5 minutes and resuspended in cell cryopreservation medium (90% freshcomplete medium, 10% DMSO), aliquoting 0.5 to 5×10⁶ cells per cryovial.The cryovials were placed at 1° C. in a freezing container, and thenmoved to a freezer at −80° C., a liquid nitrogen tank, or othersustained low temperature storage.

To thaw cells, the cryovials were removed from liquid nitrogen/storageand placed in a 37° C. water bath for 2-3 minutes until ice crystalsdisappeared. The thawed cells were then transferred to a cold 15 mlconical tube immediately using 1 ml pipette tip and the vials rinsedtwice with cold fresh medium. Ten milliliters of cold fresh medium wereadded into the 15 ml tube dropwise with gentle shaking. The cells werethen collected by centrifugation at 800 rpm (115×g) for 3 minutes, thesupernatant discarded, and the resultant cell pellet resuspended infresh medium. Cell number and viability were determined as describedherein, and then the cells were seeded into new fibronectin-coatedflasks and incubated under the conditions described above.

The morphology of feline retina-derived progenitors is shown in FIG. 1.As seen in this figure, morphology was maintained at different timepoints in the course of sustained culture. FIG. 2 illustrates a growthcurve of feline RPCs grown in two different types of cell culture media.The feline RPCs in SM senesced at passage day 10 (P10), whereas the samecells in UL continued to grow. After P14, there was an upward inflectionin growth.

The morphology of human cells in FIG. 3 shows small clusters of cellsthat were seen initially, and which were systematically converted toadherent single cell cultures by end of the first week. This wasaccomplished by dissociation during the passaging procedure as describedabove. In general, initial use of small or medium-sized clusterspromoted cellular viability, which was advantageous in the completeabsence of serum. Subsequent complete dissociation, while maintainingrelatively high cell density, can promote proliferation while avoidingdifferentiation.

The growth kinetics of hRPCs are seen in FIGS. 4 and 5. Cellsoriginating from donation were used clinically at P4. Growth seen underatmospheric oxygen is substantial, and was sustained for at least 10passages (P10), yet growth was not indefinite. Unlimited growthcharacteristics are contraindicated as they may be indicative ofpluripotency, immortalization and an increased risk of tumorigenesis.

Various serum- and xeno-free cell culture media were tested to determinethe optimum condition for propagating RPCs, including DMEM/F12(standard, Advanced and KnockOut; Invitrogen), Neurobasal (Invitrogen),Ultraculture (Lonza), and ReNcell (Chemicon). Cell culture media used inthe culture of RPCs were sometimes supplemented with N2 supplement(Invitrogen), B27 (Invitrogen or other brand), Stempro (Invitrogen),vitamin C, albumin, recombinant human epidermal growth factor (EGF),basic fibroblast growth factor (bFGF), GlutaMAX I, L-Glutamine, and/orPenicillin-Streptomycin (Invitrogen) for first 2 weeks. “SM” as providedherein refers to standard growth medium based on DMEM/F12. “UL” isreferred to herein as growth medium using Ultraculture as the basemedium. Serum-free media was used in the absence of antibiotics or withantibiotics for first two weeks, followed by antibiotic-free media forabout 6 weeks. No antifungal agents were used. FIG. 5 shows the resultsof experiments conducted to determine the optimum medium for RPCs. Forbase medium, standard DMEM/F12, a media supplemented with N2 Supplement,growth factors, glutamine or GLUTAMAX™ (GlutaMax™), was compared with“Advanced DMEM/F12”, supplemented in the same way. As seen in FIG. 6A,the “Advanced” version, which also contains supplemental vitamin C andalbumin, proved more effective and increased yield of hRPCs by 18-29%.

The effects of cell culture supplementation were also explored. To thatend, the N2 supplement was also compared with the B27 xeno-freesupplement. FIG. 6B shows that supplementation with B27 xeno-free doesincrease yield of RPCs. N2 modestly increases yield, and those amountsare sufficient for therapeutic efficacy. The effects of additionalvitamin C were also tested. A vitamin C supplement was added to theculture medium every two days. FIG. 7A shows that vitamin C does improvehRPC yield by approximately 30%.

Although Advanced DMEM/F12 contains added vitamin C, it is evident thathigher levels (0.05 mg/ml to 0.1 mg/ml) provided by additionalsupplementation are helpful for hRPC growth. Fresh vitamin C added everytwo days is sufficient; daily addition was not found to be necessary.Finally, supplementation with albumin was tested. Xeno-free humanrecombinant albumin was added to medium at 1.0 mg/ml and an enhancementof proliferation was observed (up to 27%) when added to standardDMEM/F12-based medium, but no detectable improvement was observed whenadditional albumin was added to Advanced DMEM/F12-based medium which isthe favored base medium at this juncture (and already contains addedalbumin). See FIGS. 7B and 7C.

The osmolarity of the cell culture medium was also examined by usingmedia of differing osmolarity (osm), in combination with differentcommercially available supplements. See FIG. 8. Low osm medium (KnockOutDMEM/F12; 276 mOsm/kg) was not beneficial with the commonly used neuralsupplements N2 or B27, and in some cases resulted in significantly lessyield in comparison to normal osm medium (DMEM/F12; 300-318 mOsm/kg).There were indications of a benefit for low osm if a less commonsupplement was used in combination (STEMPRO™ kit, Invitrogen, data notshown).

In addition to % viability and proliferation rate, an additional metricthat presages optimal (e.g., in vitro) outcome of initial harvest wasdeveloped, referred to herein as the “time-to-drop”. “Time-to-drop”refers specifically to the time for the dissociated cells to settle tobottom of flask in incubated growth medium. Retention in suspension(lack of drop) can be associated with cellular injury or non-viabilityresulting from the trauma of the isolation process and that successfulself-repair by cells is associated with observed ability to restoremembrane homeostasis, normal osmolarity, etc and thereby regain negativebuoyancy and thus “drop.” Delayed self-repair is stressful to cells andresults in less active/healthy cultures. Using an approximately 90% drop(based on % of population) as reporting criterion, a ˜6 hour drop timefor cells from tissue with 21.5 hour transportation time was observed,shortening to approximately 1.5 hour for cells from tissue with 4.5 hourtransport time. Cat RPC and brain progenitor cells which were platedimmediately resulted in a 1 hour drop time. Drop times of approximately1 hr or less were achievable with human cells.

Cells used to demonstrate visual improvement in human with RP werecultured under conditions of atmospheric oxygen. The ramifications ofgrowing RPCs under low oxygen conditions that more closely mimic oxygenlevels of developing fetal retina during gestation, in this case 3%oxygen (“lowOx”) were explored. As FIG. 9 shows, 3% oxygen (“lowOx”)markedly improved proliferation of hRPCs as well as more sustainedproliferation (at day 56, P10) and greatly increased overall cell yieldfrom a given donation. Growth characteristics under atmospheric oxygen(20%) are shown for comparison and reveal notably less vigorousproliferation rate as well as earlier senescence of growth (day 47, P7)and inferior total yield. FIG. 9 also shows an inflection pointcorresponding to an acceleration of growth of hRPCs in lowOx that occursat a confluence level of about 40%, emphasizing the importance of highdensity culture conditions for optimal hRPC growth. Similar results areseen in cells grown under hypoxic conditions (FIG. 10). The growthcharacteristics of WCB cells grown under hypoxic conditions werereproducible (FIG. 11).

The cells were also subjected to a steroid toxicity test, since steroidsare often used in patients at the time of transplantation. FIG. 12 showstoxicity to hRPC of the steroid triamcinolone acetonide, which iscommonly used in ophthalmic applications, but only at levels beyondanticipated clinical usage. Clinical doses of triamcinolone acetonidedid not appear to affect cell proliferation, but high doses (about >10times the expected clinical dose) may decrease donor cell viability.

Cells previously frozen in liquid nitrogen were thawed and tested forviability. FIGS. 13A-13D shows the results of a freeze-thaw experiment.These previously frozen cells were viable under both hypoxic andnormoxic cell culture conditions. Viability of the cells was found to beapproximately 94% for both oxygen conditions. Post-thawed cellsdisplayed growth kinetics similar to hRPCs maintained under continuousculture conditions.

Example 2 Characterization of RPCs Immunocytochemistry

Cells were dissociated and grown on 4 or 8-well chamber slides for 1-3days, then fixed for 15 minutes in 4% paraformaldehyde and washed 3times in PBS. The slides were blocked in a solution containing 5% donkeyserum and/or 0.3% Triton X-100 for 1 hour, followed by another PBS wash.A panel of antibodies was then incubated overnight at 4° C. to detectantigens expressed by progenitor cells. These included anti-Nestin(Chemicon 1:200), anti-vimentin (Sigma 1:200), anti-Sox2 (Santa Cruz1:400), anti-SSEA-1 (BD 1:200), anti-GD2 (Chemicon 1:100), anti-Ki-67(BD 1:200), anti-β3-tubulin (Chemicon 1:400), anti-GFAP (Chemicon1:400), and anti-GDNF (Santa Cruz 1:200). This was followed byincubation with anti-mouse Alexa 546 (Invitrogen 1:400), anti-goat Alexa488 (Invitrogen 1:400), or anti-rabbit FITC (Chemicon 1:800) secondaryantibodies. Fluorescence was detected using Leica converse microscopyand visualized by Metamorph software. Percentage positive profiles werecalculated by counting those profiles expressing specificimmunoreactivity in 6 randomly selected fields, with DAPI used todetermine total cell number.

RNA Extraction

Total RNA was extracted by using an RNeasy Mini kit (Qiagen, Calif.,USA) following the manufacturer's instructions and treated by DNase I.RNA was quantified by spectrophotometer (ND-1000; NanoDrop TechnologiesInc., Wilmington, Del.) by measuring optical density (OD) at 260 nm/280nm 1.90-2.10 and 260 nm/230 nm 1.90-2.20.

Microarray Analysis

All starting total RNA samples were quality assessed before beginningthe target preparation/processing steps by running a small amount ofeach sample (typically 25-250 ng/well) onto a RNA 6000 Nano LabChip thatwas evaluated on an Agilent Bioanalyzer 2100 (Agilent Technologies, PaloAlto, Calif.). Double stranded cDNA was synthesized from thepoly(A)+mRNA present in the isolated total RNA using the GeneChip WTcDNA Synthesis Kit (Affymetrix, Inc., Santa Clara, Calif.) and randomhexamers tagged with a T7 promoter sequence. Typically, 100 ng of totalRNA starting material was used for each sample reaction. The doublestranded cDNA was then used as a template to generate many copies ofantisense cRNA from an in vitro transcription reaction for 16 hours inthe presence of T7 RNA polymerase using the Affymetrix Genechip WT cDNAAmplification Kit. Ten micrograms of cRNA were used in a second-cyclecDNA reaction with random hexamers that were reverse-transcribed toproduce single stranded DNA in the sense orientation.

The single stranded DNA sample was fragmented (WT Terminal Labeling Kit,Affymetrix) to an average strand length of 60 bases (range 40-70 bp)following prescribed protocols (Affymetrix GeneChip WT Sense TargetLabeling Assay Manual). The fragmented single-stranded DNA wassubsequently labeled with recombinant terminal deoxynucleotidyltransferase and the Affymetrix proprietary DNA Labeling Reagent, whichis covalently linked to biotin. Following the recommended procedure,0.54 μg of this fragmented single-stranded target cDNA was hybridized at45° C. with rotation for 17 hours (Affymetrix GeneChip HybridizationOven 640) to probe sets present on an Affymetrix human-gene 1.0 STarray. The GeneChip arrays were washed and then stained withstreptavidin-phycoerythrin on an Affymetrix Fluidics Station 450(Fluidics protocol FS450_007). Arrays were scanned using the GeneChipScanner 3000 7G and GeneChip Operating Software v1.4 to produce CELintensity files.

Normalization was performed using the probe logarithmic intensity error(PLIER) estimation method, which includes a quantile normalizationprotocol within the associated software algorithm. Briefly, the probecell intensity files (*.CEL) generated above were analyzed usingAffymetrix Expression Console software v1.1 using the PLIER algorithm togenerate probe-level summarization files (*.CHP). The algorithm used wasfrom PLIER v2.0 (quantification scale: linear; quantification type:signal and detection p value; background: PM-GCBG; normalization method:sketch-quantile). Microarray data was then evaluated using JMP Genomics4.1 (SAS Americas). The data was analyzed by one-way ANOVA with a posthoc t-test and the resulting p-values corrected using an FDR a<0.05. Theresulting data table was annotated. The JMP software was also used togenerate a Principal Component Analysis, a Venn diagram, as well as ahierarchical cluster and heat map, using the default fast Ward's method,in addition to volcano plots from the ANOVA results.

Real-Time qPCR Assay

Selection of candidate markers was based on the results of previous workwith cells of this type, together with potential relevance to thecurrent study. Particular emphasis was placed on markers associated withimmature cells of neural lineage, as well as selected markers for neuraland glial differentiation. Two micrograms of total RNA from the samplepreparation was reverse transcribed with Omniscriptase ReverseTranscriptase kit (Qiagen, Calif., USA) and 10 μM random primers (Sigma,Mo., USA) according to the manufacturer's instructions. Quantitative PCRwas performed using a 7500 fast Real-Time PCR System (AppliedBiosystems, Irvine, USA) using Power SYBR green (Applied Biosystems,Irvine, USA) or Taqman gene expression assay (Applied Biosystems).

Resolution of the product of interest from non-specific productamplification was achieved by melting curve analysis when using the SYBRgreen method. β-Actin or GDNPH were used as endogenous controls tonormalize gene expression. The following general real-time PCR protocolwas used: denaturation program (95° C. for 10 minutes), quantificationprogram (95° C. for 15 seconds and 60° C. 1 30 min) repeated 40 cycles,melting curve program (95° C. 15 sec and 60° C. 1 min with continuousfluorescence measurements), and finally a cooling program at 40° C. Eachreaction was performed in triplicate. Graphs were plotted and analysiswas performed by the ΔΔC_(t) method (7500 Fast system software 1.4 andDataAssist 2.0, Applied Biosystems, Irvine, USA) and JMP software 4.1(SAS Americas). All data points are expressed as mean±standard Error(SE). Statistical difference was determined using t-test. Data wereconsidered significant when p<0.05.

Cytotoxicity Study

Cell Counting Kit-8 (CCK-8; Dojindo Molecular Technologies Inc.,Gaithersburg, MD) was used to determine cytotoxicity of RPCs. The kituses WST-8, which, upon bioreduction in the presence of the electroncarrier 1- methoxy PMS, produces a water-soluble colored formazan.Ninety-six well plates containing 90 μl of cell suspension per well wereinoculated with 10 μl of CCK-8 pre-packaged solution. The plates wereincubator for 2 hours and the OD₄₅₀ of the supernatant was measured.Each experiment was performed in quadruplicate on at least threeseparate experiments.

Feline RPC cells stained by ImmunoCytoChemistry (ICC) markers showedhigh levels of expression of vimentin. See FIG. 14. The same wasobserved in human RPCs. Lineage markers including nestin, vimentin,Ki-67, β3-tubulin, glial fibrillary acidic protein (GFAP), and rhodopsinrevealed the presence of neurons, photoreceptors, glia and demonstratethe retained multipotentiality and heterogeneity of RPCs cultures. Thefeline RPC genetic profile was observed over time using qPCR of markertranscripts. Dynamic changes in expression profile: general trend ofdownward quantitative changes in marker expression with time in culture,as also seen with human RPCs. See, e.g., FIG. 15. Gene expression wasalso compared between UL and SM cell culture media conditions by qPCR(FIG. 16A). Because proliferation of feline progenitors (both RPC andBPC) was not sustained well in SM cultures, an investigation was madeinto comparative gene expression. With SM day 13 used as baseline forcomparison (set to “1.0”), feline RPCs grown in SM and UL were comparedat culture Day 31. Most markers examined showed decreased expressionover time in culture, however the SM culture showed marked elevation inGFAP expression, consistent with progressive loss of multipotency andtendency toward restriction along the glial lineage. Cells in UL mediadid not show this.

Feline RPC versus brain progenitor cells (BPCs) were also compared. SeeFIG. 16B. Feline RPCs showed relatively increased expression of somemarkers relative to BPCs; these included Dachl, Lhx2 and Pax6, which aretranscription factors involved in retinal specification and development,as well as the transcription factors Hes1 and Hes5, also involved inretinal development. CD133, nestin, Sox2, vimentin are general CNSprogenitor markers, while β3-tubulin, Map2, and PKCα are lineagemarkers.

Feline RPCs were transplanted to the subretinal space of dystrophicAbyssinian cats and isolated post-transplantation cells subjected tostaining. See FIG. 17. The cells survived transplantation to thesubretinal space and in addition showed the ability to migrate into therecipient retina. Engrafted cells showed an ability to differentiateinto what appear to be Mueller glia, both morphologically and byvimentin labeling. Mueller cells are glia that are specific to theretina and that extend across the full thickness of the retina toprovide structural stabilization. They are important to numerous retinalfunctions, including neuronal survival.

The morphology of human RPCs using marker expression by ICC wasdetermined. FIGS. 18 and 19 show the percentage of expression of certainkey markers within the cultured population. Nestin expression wasconfirmed and is believed to be associated with neural stem/progenitorcells and RPCs. Vimentin was very heavily expressed by this population,although its expression was believed to be non-specific to RPCs. Sox2 istranscription factor also associated with neural development and wasalso present in hRPCs. SSEA-1 (also known as CD15, LeX) has beenassociated with pluripotency in ES cells; studies have showed expressionof this marker by subset of multipotent brain and retinal progenitors(RPCs), which are not pluripotent. Expression of GD2-ganglioside wasalso seen in hRPCs. Ki-67 is marker of active proliferation and was usedto confirm that the cells of this embodiment are proliferative andmitotically active at the time of clinical use. The presence of thismarker distinguishes hRPCs from populations of post-mitotic precursors(Surani, M. A. and McLaren A. (2006) Nature 443(7109): 284-285). Ki-67in the relative absence of OCT4 expression distinguishes RPCs frommitotically active pluripotent stem cells (ES, iPS), which are unsafefor transplantation unless subjected to further differentiation. Thelevel of Ki-67 activity also allowed monitoring of the quality (healthand suitability) of the isolated hRPC cultures. β3-tubulin is a markerof neuronal development and was found to be expressed at moderate levelsin the cultures, suggestive of neural lineage determination and thepotential for differentiation into neurons. Since neuron formation tendsto be lost at high passage numbers, expression of this marker mayconfirm the retention of multipotency by the cultures. GFAP is a markergenerally associated with glial differentiation, particularlyastrocytes, although it is also expressed by retinal Mueller cellsfollowing a variety of perturbations or in culture. Again, the lowpercentage of GFAP expression seen is suggestive of the rate ofspontaneous differentiation into glial cells and helps us confirm theretention of multipotency by the cultures, however GFAP is also known tobe expressed by immature progenitors as well. GDNF is a neurotrophicfactor associated with rescue of neurons, including photoreceptors, insome animal models and the hRPC populations isolated as described hereincan express this factor by ICC, however negative ELISA data indicatethat the factor is not necessarily secreted. Additional factors arelikely to be more important to RPC-mediated photoreceptor rescue.

Notably, hRPCs can be distinguished from fibroblasts (hFB) as shown bymarker expression at the RNA level. See FIG. 20, which shows a qPCR heatmap demonstrating that vimentin is a marker that is very highlyexpressed by hRPCs but not fibroblasts, and additionally reveals dynamicchanges in relative profile expression with time in culture. FIGS.21A-21C show the results of a qPCR experiment comparing hRPC versus hFBand expanding marker detection. Multiple genes are identified that arehigher in RPC (AQP4, CD133, GFAP, MAP2, MASH1, nestin, Notchl,recoverin, SIX6, SOX2, and 1 (KLF4) that is lower (therefore higher inhFB). Expression levels vary with time in culture, but the relativepredominance between cell types appears fairly consistent. FIG. 22contains a list of 11 genes (from the profile of approximately 26 used)that exhibit consistent behavior (in hRPC vs. hFB) between differentdonations. Genes consistent across all 3 donations are highlighted inyellow, and are as follows KLF4 (RPC<FB); and GFAP, MAP2, nestin,recoverin, SIX6, and SOX2 (RPC>FB).

The expression levels of selected markers were tracked as a function oftime in culture by real time qPCR. As shown in FIG. 23, highly expressedmarkers tend to ease off of peak expression levels with time. Forexample, Ki-67 tends to drop with time, consistent with progenitorstatus and lack of immortalization. MHC and GDNF expression show amodest increase over time in culture. Changes in the expression level ofmarkers in early vs. late passage cells were tested, the results ofwhich are shown in FIG. 24. Cell cycle genes and Six6 (a transcriptionfactor critical to retinal development) are most strongly down-regulatedin late passage cells. Conversely, the neuroprotective factor GDNF tendsto be up-regulated at late passage, possibly as a result of cellularstress.

A summary of microarray data that distinguishes (human) RPCs from neuralstem cells (BPCs) is shown in FIG. 25. Principal component analysisshows clear segregation of RPC data sets (3) from BPCs (3),demonstration that RPCs as a cell population type can be distinguishedfrom analogous brain-derived cell types, such as BPCs, based upon theirtranscriptome. A Volcano plot shows the basis of this difference in theform of close to 1000 transcripts significantly up-regulated in RPCscompared to BPCs, as well as ˜600 transcripts that are significantlydown-regulated. The comparison is further delineated using dendrogramsfor transcripts according to specific gene categories and specific genesare identified. For instance, the transcription factor BHLHE41 (basichelix-loop-helix family, member e41) is highly expressed by RPCs withvery low expression by BPCs. This gene encodes a transcription factorthat belongs to the Hairy/Enhancer of Split subfamily of basichelix-loop-helix factors. The encoded protein functions as atranscriptional repressor. Other transcription factors preferentiallyexpressed by RPCs over BPCs include HHEX, SOX3 and SOX13, HOXB2, LHX2,KLF10, TLE4, MYCBP, TFAP2A, FOSL1 and 2, FOXD1, NHLH1, GBX2, NEUROD,MET, etc. In terms of signaling molecules, numerous transcripts areshown to be expressed by RPCs at markedly higher levels than inBPCs/neural stem cells, including for WNTSA and B, KDR, LIF, CALB1,RGS4, CAV2, IL11, IL1R1, IL1RAP, IL4R, IL21R, CXCL6 and 12, CXCR7, DKK1,HBEGF, SMAD7, BMP2, etc. Similarly, the ECM matrix genes fibronectin,LUM, ALCAM, TGFBI, ECM1, PARVA, as well as the collagens: 4A1, 4A2, 5A1,5A2, 7A1, 9A2, 13A1, 18A1, and a variety of other ECM genes are alsopreferentially expressed by RPCs.

Experiments testing marker expression after differentiation withretinoic acid (RA) in hRPCs were conducted; the results are shown inFIG. 26. The genetic profile of hRPCs grown in standard proliferationmedium (SM) were compared to RA-based differentiation conditions (SMwithout GFs+RA). The proliferation marker Ki-67 is decreased, as isvimentin, while the tumor suppressor gene p21 goes up, along withlineage markers like AIPL-I, MAP2, NRL, CRALBP, GFAP and recoverin. Theincrease in both glial and neuronal markers is consistent withmultipotency of the cultured RPC population. Other markers that can beused to practice the invention to identify hRPC cells are described inU.S. Pat. No. 7,419,825.

In addition, gene expression of hRPCs grown under lowOx conditions incomparison to atmospheric Ox were tested (FIG. 27). The data indicatesthat there are detectable changes in gene expression, with upregulationof the surface markers CD9 and CD73, but mostly down-regulation of geneswas observed, including the tumor suppressor gene p21 as well as thelineage markers CRALBP, GFAP, MAP2, NRL, and recoverin. These changesare most consistent with decreased expression of non-essential genes asthe cells proliferate in a sustained manner under these permissiveconditions. GDNF is also down regulated, perhaps because of decreasedneed for autocrine neuroprotection.

Differences in gene expression between hRPCs derived from differentdonors and cultured in a variety of cell culture media conditions andtime points were tested and the results are shown in FIGS. 28-30. FIG.28A shows the results of an experiment that detected gene expression byqPCR at SM conditions at different time points; FIG. 28B illustratesgene expression by qPCR at SM-UL (initial UL, then SM) conditions atdifferent time points; FIG. 29A illustrates gene expression by qPCR atSM-FBS (initial SM+FBS, then SM) conditions at different time points.FIG. 29B illustrates gene expression by qPCR with SM alone, 2 differenttime points. Most of the tested markers showed decreased expression overtime in culture, while some remain roughly level. Notably, of the testedmarkers, only GDNF expression increased over time in culture. FIG. 29Cillustrates gene expression by qPCR with SM (after initial SM+FBS) same2 time points. As shown in FIG. 29B, only GDNF is elevated. FIG. 30represents a summary of these time point comparisons.

qPCR was used to further characterize hRPCs in relation to growth factorpathways and was directed at relative expression of secreted factors.Specific panels included angiogenesis and WNT signaling pathways. Cellsused in these experiments were derived from hRPCs of low passage thatwere grown under normoxic (20% oxygen) and hypoxic (3% oxygen)conditions; from a working cell bank, which includes hRPCs of higherpassage that were grown under hypoxic conditions; or from human fetalretinal tissue at Day 0 (the day of donation), which represents thebaseline origin of hRPCs. Human fetal RPE (hRPE) cells and human fetalfibroblasts (hFB) were used for comparison.

FIG. 31 is a heat map analysis of qPCR data for growth factor pathwaystudy. Each vertical column is a different cell type or treatmentcondition. Human fetal RPE and fibroblast cells were used as comparators(first 2 columns), the 3 columns to the right are hRPCs. One cytokinewith notably high expression in 2 of 3 hRPC banks, plus intermediate inthe other, yet not expressed by RPE or FB is SPP1 (osteopontin, OPN),which is currently a primary candidate for trophic mechanism of actionfactor. Another candidate is PTN (pleiotrophin), which is also highlyexpressed, although this factor appears to be less specific than SPP1(OPN). Other potential candidates from this data in terms of expressionlevel are MDK (midkine), TGFB1 (TGFβ1), JAG1, VEGFA (VEGF A), PGK1(phosphoglycerate kinase 1) and cases can be made for others to varyingextents such as GDF11, DKK1, PPIA (peptidylprolyl isomerase A), and LIF,etc., depending on criteria used. Also, B2M (β2-microglobulin, acomponent of MHC class I) is not a cytokine per se, but is stronglyexpressed by hRPCs and a component of MHC class I.

When specificity is emphasized, the dendrogram clustering shows thatthose listed from HBEGF (heparin-binding EGF-like growth factor,HB-EGF), JAG1, down to SPP1 (OPN) all show a general specificity forhRPCs over fetal hRPE and fetal hFBs, and therefore could contribute toa heterogeneous “cocktail” effect. This includes PTN, as well as lowlevels of IL1B (interleukin 1β). It is also possible that the 20% MCBhas the maximal trophic efficacy in which case the factors listed fromMDK down to LEFTY2 would be of interest. UBC (ubiquitin C) was alsodetected. A number of these genes are known to play a role in retinaldevelopment, including GDF11, lefty, nodal, DKK1, LIF, etc. Many areknown to be neurotrophic, including SPP1, NTF3, HB-EGF or related toneural development such as midkine, neuregulins (NRG1, 3), the JAGs,etc.

Another view of qPCR data from the growth factor pathway study isprovided in FIG. 32, seen here as a histogram. Human fetal RPE cellswere used for comparison. Viewed quantitatively in terms of expressionlevel, secreted genes that showed particularly high expression by hRPCsincluded FGF9, GDF10, IL-1A (interleukin 1 alpha), PTN, and SPP1(osteopontin, OPN). All of these genes were found to group in whatappears to be a relatively hRPC-specific cluster of the heat map, above,and so are considered candidates for mediating a trophic mechanism.Other genes showing lower relative expression, but still elevated,include BMP2, FGF7,13,14, Lefty1,2, nodal, NTF3, thrombopoietin andpotentially VEGF A,C. Genes that were preferentially downregulated byhRPCs relative to hRPE were JAG2, NGF, inhibin beta B (INHBB), andIL-10.

RPC are active before and during the period of retinal vasculogenesisand as such can be expected to play a role in angiogenesis, especiallyas they begin to differentiate. Factors and receptors involved inmolecular pathways regulating angiogenesis could also be potentiallyneurotrophic in the setting of the degenerating retina. Also, activationor inhibition of these pathways could be important in a range of retinalconditions, including AMD, either beneficially or as an undesired sideeffect.

All genes shown in FIG. 33A were upregulated compared to RPE by at least10 fold. Genes upregulated more than 100 fold included the VEGF receptorKDR, chondromodulin/LECT1 (hypoxic condition only), the transcriptionfactor PROX1, and the receptor TEK (TIE2). The factor with the highestexpression relative to RPE was pleiotrophin (PTN) at over 10,000 foldfor hypoxic hRPCs and approaching that for normoxic cells.

Clear evidence of increased expression of angiogenesis-related genes andadditional confirmation of high levels of PTN expression, one of the toptrophic factor candidate genes, were detected. Elevated expression ofthe surface markers KDR and TEK, both identified in previous screens,were also confirmed here. The VEGF receptor KDR was consistently foundto be elevated in RPCs versus other cell types. The microarray data alsoshowed KDR expression in hRPC>hBPC (brain progenitors) of 80 fold, andin porcine RPCs>BPCs of 106 fold. KDR is therefore a surface markerpotentially useful for identification and enrichment of RPCs, is a wayto distinguish RPCs from BPCs (neural stem cells) and is of likelyimportance to the function of RPCs.

WNT pathways are believed to be important to neural development,including the differentiation of neurons and glia, throughout the CNSand including the retina. Genomic studies have previously identifiedconsiderable evidence of WNT pathway activity in hRPCs, based on geneexpression levels of WNT-related genes, including WNTs, “frizzled”receptors, and WIF (WNT inhibitory factor). In FIG. 33B, all genes shownwere upregulated compared to RPE by at least 10 fold. The genesupregulated by more than 100 fold included FRZB, SFRP4 (normoxic), TLE2(hypoxic only), and WNT7B (normoxic). SFRP4 (hypoxic) and WNT7B(hypoxic) were both upregulated at levels greater than 1000. Justreaching 10,000 fold change was WIF1 (hypoxic only) showed ˜10,000 foldchange in expression.

In summary, clear evidence of WNT pathway gene expression, includingprominent expression of WNT inhibitory genes, was observed by microarrayanalysis. Prior microarray data already showed that WIF1 ispreferentially expressed by hRPC>hBPCs (brain) by 45 fold. Markedupregulation of SFRP4 and especially WIF1 (both of which can result fromFRZB activation) appears to be characteristic of hRPCs grown underhypoxic conditions. This relates to the ability of hRPCs to bettermaintain an immature state and proliferate for extended periods underhypoxic conditions, thereby hugely increasing the yield of thesedifficult to grow cells. The qPCR data presented above intersectmeaningfully with prior results obtained from microarray. These showedthat LIF is preferentially expressed in hRPC>hBPC (brain) by 65 fold,HB-EGF by 30 fold, DKK1 (dickkopf 1) by 23 fold, osteopontin (SPP1, OPN)by 6 fold, TGF beta 1 and BMP2 by 5 fold, and JAG1 by 3 fold. Each ofthese genes contributes to the composition/identity of hRPCs vis-a-visthe analogous brain stem/progenitor cells (neural stem cells“), as doesKDR to an even greater extent (80 fold) and also WIF (45 fold).

Principle component analysis (PCA) data from the microarray data showsthe ability to distinguish cellular populations (therapeutic,non-therapeutic/control) based on global gene expression patterns. Thisalso pertains to characterization of hRPCs by showing how closelyrelated the samples are and the extent to which age (time in culture)and culture conditions (normoxic versus hypoxic) influence expressionpatterns.

Three pairs of fetal eyes (biological replicates) were obtained on thesame day and provided RNA at Day 0 (retinal tissue) and were also usedto grow RPCs (normoxic and hypoxic), as well as scleral fibroblasts. RNAwas later extracted from the cultured cell populations and all sent forsimultaneous microarray analysis.

FIG. 34A depicts the PCA data, which clearly shows that the culture RPCsdiffer in gene expression from the fetal retinal tissue from which theywere derived, but also from scleral fibroblast. RPCs are closer to fetalretina than are the fibroblasts. In addition, normoxic and hypoxic RPCcan be distinguished, although they appear to form a continuum, withyounger cells at one end and older at the other. In FIG. 34B, hRPCs areclearly distinguished from P0 retinal tissue of origin.

The influence of time in culture is evident in that the oldest cells(hypoxia WCB) can be distinguished from the other younger samples. Thedifference between normoxic and hypoxic conditions is not showing strongsegregation.

A whole genome microarray study was undertaken to characterize thecomposition of hRPCs relative to other cells types, including how theydiffer from tissue of origin (fetal retina) and from grossly derangedand dangerous tumorigenic analogs, i.e., retinoblastoma (RB). Also, thestudy was undertaken to delineate the similarities and differencesbetween normoxic and hypoxic hRPC cultures. Global gene expression ofcell populations was compared on Affymetrix human gene chips.

Principle component analysis (PCA) provides immediate three-dimensionalvisualization of the global similarities and differences between cellsample populations. See FIG. 34C. The grouping together of similarsamples (all same color, all in triplicate) demonstrates the reliabilityof the data. Clear separation between fetal retinal tissue (”retina“)was seen compared to the 4 different hRPC samples. The RPCs differedsomewhat between each treatment condition, yet they segregate away fromthe other ocular cell types tested, i.e., fetal RPEs, fetal FBs, and RB.A line can be drawn to separate neural retina and neural retina-derivedcells (left) from non-neural retinal cells (upper right). Finally,normoxic and hypoxic hRPCs are relatively close to each other and,although it may be possible to distinguish them to some extent, theyappear to be closely related. The data shown are consistent with priordata regarding the relative similarities/differences of these differentcell types. FIG. 35 shows cluster analysis of 3 hRPC populations (inorder: hypoxic MCB, normoxic MCB, hypoxic WCB) versus fetal retinaltissue (triplicates used for each population). FIG. 35 is a Volcano plotshowing the results of a comparison experiment between hypoxic MCB andtissue. The data show that hRPC populations are distinguishable from theoriginal tissue populations. With few exceptions, genes that are seen asred in the tissue (right column) are green in the RPCs, and vice versa.

FIG. 37A shows the number of genes expressed differently between hRPCgroups as a function of treatment conditions (fetal retinal tissue usedas comparator). The majority of differences are accounted for by changesversus tissue (11,706, in center, gray), which are shared among RPCpopulations, while individual overlap and differences are seen aroundthe periphery of the diagram. Each hRPC population expresses betweenapprox 1300-2100 distinct genes. The number of genes expresseddifferently between hRPC groups was measured as a function of passageand treatment condition. See FIG. 37B. Cells grown under normoxicconditions were also compared. The hypoxic MCB differs less from thenormoxic MCB (same passage number) than does the hypoxic WCB, which isat a later passage number. Thus, time in culture does have ademonstrable influence on gene expression in hRPCs, although it is farless than between cell types, or between hRPCs and tissue of origin.

FIGS. 38A and 38B are Volcano plots showing different hRPCs versustissue of origin, whereas Volcano plots in FIGS. 39A and 39B showhypoxic hRPCs versus normoxic. “Volcano” represents a style of plotshowing each gene as a data point, plotted relative to fold change (upor down, X axis) and statistical significance (function of p-value, Yaxis), providing an overview of how many genes are changing, how muchand in which direction (up vs. down). The results indicate that a largernumber of genes consistently change going from tissue to hRPC, thanbetween hRPC conditions. In the former, more genes are downregulatedthan upregulated, presumably because more differentiated cell typespresent in the tissue are lost or overgrown in culture by moreprimitive, proliferative types. However, when comparing hypoxic tonormoxic, more genes are upregulated in hypoxic conditions versusnormoxic.

Specific genes and pathways were examined by microarray analysis aswell. hRPCs, fetal retinal tissue (Day 0), and human fetal fibroblasts(normoxic) were compared. Adrenomedullin was upregulated in hRPCs vs.tissue. Pleiotrophin was found to be upregulated in hRPCs vs. tissue andhFB. Osteopontin was upregulated in hRPCs vs. hFB. Angiogenesis pathwayswere also found to be upregulated. Angiopoietins generally elevated inhRPCs vs. tissue and in hFB to lesser extent. ANGPTL4 is 30-60 foldupregulated in hRPCs vs. tissue and ˜20 fold vs. hFB. BAI3 (inhibitor)is downregulated approximately 30-fold decreased in hRPCs vs tissue.Thrombospondin 1 is upregulated ˜100-fold in hRPCs vs. tissue whilethrombospondin 2 is upregulated ˜40 fold in hRPCs vs. tissue. Matrixmetallopeptidase 1 is ˜200 fold upregulated in hRPC vs. tissue. Theadhesion molecule NCAN (neurocan) is 16-21 fold downregulated in hRPCvs. tissue. The oncogenes/proliferation-associated genes MYC, MYCN, andNBL1 were also measured. MYC is upregulated 8-9 fold in hRPCs vs.tissue, consistent with proliferation of hRPCs. MYCN (v-myc related,neuroblastoma) is 25-40 fold downregulated in hRPCs vs tissue. NBL1 isdownregulated 3-4 fold. A wide range of mitochondrial/metabolic genesincluding ATP synthase H⁺ transporters and solute carrier family 25,were all generally upregulated 2-4 fold in hRPCs vs. tissue, howeverSLC25A27 (member 27) was ˜20 fold downregulated. These data areconsistent with the concept that cultured hRPCs are more metabolicallyactive and more proliferative than cells in fetal retinal tissue oforigin.

Growth factor expression was also examined. CTGF (connective tissuegrowth factor) is strongly (>70 fold) upregulated in hRPCs vs. tissue.LIF (leukemia inhibitory factor) is similarly upregulated in hRPCs vs.tissue. BDNF and EGF are moderately (2-14 fold) upregulated in hRPCs vs.tissue. CNTF 15 downregulated ˜25 fold in hRPCs vs. tissue. FGF9, whichis a candidate trophic factor based on prior studies, is confirmed to beupregulated (4-6 fold) in hRPCs vs. hFB, and yet is 14-25 fold decreasedvs. tissue. FGF5 is the family member most strongly upregulated in hRPCscompared to tissue (20-40 fold), yet expression is lower vs hFB. FGF14is the most downregulated vs tissue, by 16-38 fold. HGF (hepatocytegrowth factor) is 30 fold downregulated vs. hFB. Members of theinsulin-like growth factor binding protein family, i.e., IGFBP3, -5, and-7, are generally upregulated in hRPCs vs. tissue. Members 3,5,7 are allstrongly upregulated (15-110 fold). IGFBP3 and 5 are most stronglyupregulated, specifically in the hypoxic cells (MCB, WCB). NTF3, acandidate factor, was not elevated over tissue, but was 8 fold elevatedin normoxic hRPCs vs. hFB. NTRK2 (neurotrophic tyrosine kinase,receptor, type 2) was modestly upregulated in hypoxic hRPCs vs. tissueand strongly upregulated in all hRPCs vs. hFB (50-180 fold). PDGFC(platelet derived growth factor C), another candidate trophic factor,was upregulated 20 fold vs. tissue. VEGFA (VEGF A) was modestlyupregulated by hypoxic hRPCs. DACH1 was modestly higher vs. hFB, butstrongly downregulated in hRPCs vs. tissue. DLG2 (a synaptic marker) wasalso strongly downregulated in hRPCs vs. tissue. KLF family membersshowed downregulation of KLF4 vs. hFBs (3-4 fold), and upregulation ofKLFS vs. tissue (4-5 fold).

NeuroD1 (transcription factor associated with differentiation) was verystrongly downregulated vs tissue (>300 fold), as was NeuroD4 andneurogenin 1 (and 2), as well as neuronatin (2-10 fold). NOG (noggin), afate specification-associated factor is strongly expressed by hRPCs(10-21 fold) vs. tissue. OTX2 (ocular transcription factor) was verystrongly downregulated in hRPCs vs. tissue. PAX6, SIX3, SIX6, RAX, RAX2(ocular transcription factors) were only modestly downregulated in hRPCsvs. tissue. PAX6, at least, is known to be expressed by at least someRPCs.

DCX (post-mitotic neural blast marker) and RELN (reelin) a migratoryneuroblast marker were strongly downregulated vs. tissue (the latter inhypoxic hRCs). SOX2, an important neurodevelopmental transcriptionfactor, was unchanged vs tissue, but very strongly upregulated in hRPCsvs. hFB (>100 fold). Basoon, a mature retinal synaptic marker andrecoverin (mature retinal cell marker) were strongly or very stronglydownregulated vs tissue (20 fold and 50-450 fold, respectively).

CD44, surface glycoprotein, was upregulated 6 fold in hRPCs vs. tissue.CCL2 chemokine (MCP-1) was strongly upregulated (68-105 fold) in hRPCsvs. tissue. CXCL12 (SDF-1) is the ligand for the (nonspecific) hRPCsurface marker CXCR4. SDF-1 is downregulated compared to hFBs, soexpression is low (as we know from ELISA), however, it is elevated 4-12fold in hRPCs vs. tissue. CXCR4 is a surface marker of hRPCs and wasclearly upregulated in hRPCs vs. hFB. Comparison to tissue wasinteresting in that normoxic MCB was lower than tissue, but the hypoxiccells similar. This confirms that both hRPC conditions express CXCR4,with hypoxic expressing more, and retinal tissue similar to hypoxiccells.

IL-11 and IL-18 were upregulated vs tissue. IL-lA (IL-1a) wasupregulated vs. tissue, although “not” for hypoxic MCB. A number of ILreceptors were upregulated vs. tissue, most prominently IL-7R (60-105fold), IL-31RA (28-80 fold), and IL-4R (18-40 fold). These all representpotential positive markers for hRPCs vs tissue of origin.

WNT pathway genes upregulated vs tissue included some previouslyidentified by qPCr, including WNT7B, and SFRP4. Also upregulated inhRPCs vs tissue were DKK2, FZD6 SFRP1, WNTSA. WIF1 was stronglyupregulated vs hFB (for hypoxic), yet strongly downregulated vs tissue(for normox). Notch pathway genes were downregulated or unchanged vstissue except Jagl, which is candidate hRPC trophic marker, which wasupregulated (8-13 fold). The most strongly downregulated were DLL4 andHEYL.

JAK-STAT genes were moderately but uniformly downregulated vs hFB (2-5fold), except STAT3 which was unchanged for hypoxic vs. FB. They wereunchanged vs. tissue. Apoptosis genes were mixed. Most elevated vs.tissue was GADD45B. More prominently GADD45G and DAPL1 weredownregulated (20-56 fold). BMP2 (candidate) was upregulated vs hFB, butno change seen vs. tissue. Other BMPs were generally decreased vs. FBand also unchanged vs. tissue. TGF beta genes were unchanged throughout.

HIF1A (HIF1alpha) was moderately decreased vs. tissue (4 fold).Neuropilin 1 and especially 2 appear to be increased (except for hypoxicMCB). RICTOR was modestly decreased vs. tissue (2 fold). Toll-likereceptors 3-7 and 9 were unchanged vs. tissue. DCX was stronglydownregulated vs. tissue, as were a number of other neural markers to alesser degree, the next most being NRXN1 (neurexinl, >25 fold). GFAP wasclearly upregulated vs. hFB, less clearly vs. fetal retinal tissue. Alarge number of retina-associated genes are strongly downregulated byhRPCs vs. fetal retinal tissue of origin. These include CRX, EYS,IMPG1,2; NRL, recoverin, RGR, RP1, and VSX1, 2. Of 98 small non-codingRNAs (SNORDs) examined, almost all were downregulated vs. tissue,sometimes massively, except 114-6, 49B, 75, and 78, with 114-2, 44, 49A,74, 79, 96A being suggestive but somewhat inconsistent acrossconditions. Regardless, upregulation was not to a high level. Insummary, the data indicates that CTGF is an hRPC marker and may behelpful to determine the trophic mechanism underlying therapeuticefficacy. Other such markers include SPP1 (OPN), PTN, LIF, FGF9, JAG1,IL-1A, IL-11, IL-18, and noggin. NTRK2 neurotrophin receptor is a hRPCsurface marker, as are the interleukin receptors IL7R, IL-31RA, andIL-4R. Notably, HGF appears to have potential as a negative marker forhRPCs, or alternatively, in a method for detecting contaminating celltypes. Downregulation of the ocular transcription factors DACH1, OTX2,CRX, NRL, VSX1,2 and differentiation transcription factors NeuroD1 (and4), the post-mitotic blast marker doublecortin (DCX), synaptic markerDLG2, many notch pathway genes, neurexinl, as well as the mature retinalmarkers recoverin and interphotoreceptor matrix genes IMPG1,2 are alsouseful to distinguish cultured hRPCs from tissue of origin. Cellsexpressing those markers are more specified, mature, and lessproliferative and therefore far less plentiful than in proliferativehRPC cultures. CPA4 is highly upregulated, while PAR4 and a long list ofSNORDs are downregulated.

Similar data was obtained for feline RPCs. In FIG. 40, a tabular summaryof experiments testing cell culture conditions over time is provided.FIGS. 41-44 show the results of an experiment measuring gene expressionin feline RPCs by qPCR in UL media at the various time points. Notably,in UL media, the tested markers were downregulated, with the progenitormarkers nestin and vimentin showing elevated expression over Day 0baseline. Cyclin D2 is elevated in UL at time points after Day 0baseline. The pattern of changes in expression within the testedprofiles for feline cRPCs is presented as a radial graph in FIG. 45.Genes having high copy number are toward the center of the graphs, whilegenes having lower expression are peripheral. Progenitor markers arelisted from nestin at 12 o'clock clockwise (on the radial graph) tovimentin at about 6:30. Lineage markers are also shown. Note thatexpression tends to be highest across markers at Day 0 (dark blue) anddecrease for some, but not all, markers with time. The biggest decreasein expression appears to occur in going from Day 0 to the first timepoint in culture measured (Day 31). Decreased expression is seen in asubset of both lineage and progenitor markers. A chart summarizing theexpression data for feline RPCs is presented in FIG. 46, representingqPCR data across different donors and culture conditions.

An enzyme-linked immunosorbent assay (ELISA) was carried out for thepurpose of characterizing hRPCs and potential mechanism of action (i.e.,neuroprotection). These studies were performed by either multiplex assayor sandwich ELISA. For multiplex ELISA assay, Assay Buffer was added toa 96 well filter plate and the plate placed on a shaker for 10 minutes.The plate was then cleared by vacuum and 25 μl of Assay Buffer or otherappropriate buffer added to each well with 25 μl ofstandard/sample/control added to the appropriate wells. Then 25 μl of amixture containing requested cytokines (1:50 dilution) that wereconjugated to beads was added. The plate was then placed on a shaker, at4° C. overnight. The plate was then washed 3 times. Detection antibodywas added and the plate placed on a shaker for one hour at roomtemperature. Then 25 μl of Phycoerythrin (1:25 dilution) was added toeach well on the shaker for 30 minutes. The plate was then washed threetimes and 150 μl of Sheath Fluid is added to each well. The plate wasthen read using a Luminex 100 reader and Softmax Pro software. The datawas calculated using Millipore's BeadLyte Software.

For sandwich ELISA, markers were measured by two-antibody ELISA usingbiotin-strepavidin-peroxidase detection. Polystyrene plates were coatedwith capture antibody overnight at 25° C. The plates were washed 4 timeswith 50 mM Tris, 0.2% Tween-20, pH 7.0-7.5, and then blocked for 90minutes at 25° C. with assay buffer. The plates were washed 4 times and50 μl assay buffer was added to each well, along with 50 μl of sample orstandard prepared in assay buffer. The plates were then incubated at 37°C. for 2 hours. The plates were washed 4 times and 100 μl ofbiotinylated detecting antibody in assay buffer was added and incubatedfor 1 hour at 25° C. After washing the plates 4 times,strepavidin-peroxidase polymer in casein buffer (RDI) was added andincubated at 25° C. for 30 minutes. The plates were washed 4 times and100 μll of commercially prepared substrate (TMB; Neogen) was added andincubated at 25° C. for approximately 10-30 minutes. The reaction wasstopped with 100 μl 2N HCl and the A450 (minus A650) was read on amicroplate reader (Molecular Dynamics). A curve was fit to the standardsusing a computer program (SoftPro; Molecular Dynamics) and cytokineconcentration in each sample was calculated from the standard curveequation. Data reflects protein secretion from averaged samples.Expression of five candidate trophic factors, GDNF, BDNF, VEGF, OPN, andSDF-1, was evaluated under standard normoxic (20% O₂) as well as lowoxygen (3% O₂) conditions.

In FIG. 47, GDNF expression was found to be undetectable at level ofsecreted protein (<0.1 picogram/ml). BDNF expression was out of range(OOR) to the low side, but just detectable at average concentration of1.58 pg/ml for normoxic conditions and 0.55 pg/ml for hypoxicconditions. VEGF was OOR to low side for the normoxic (20% O₂)condition, but detectable for hypoxic (3% O₂) at average concentrationof 92 pg/ml. OPN (osteopontin, SPP1) expression was strongly positive(nanogram range instead of picogram) under both conditions,approximately double for norm ox versus low ox (average=72.2 ng/ml vs31.3 ng/ml, respectively). Those are notable levels in both instancesand suggest a role for this known neuroprotective/anti-apoptotic factor.SDF-1 (stromal cell-derived factor 1) was OOR to the low side, butdetectable with average concentration of approx 48 pg/ml for both normox and low ox cultures.

Osteopontin (OPN, SPP1) is highly expressed by hRPCs and secreted intothe surrounding media at what are predicted to be physiologicallysignificant concentrations.

Other expression data showed differential expression of this gene versusother cell types. Taken together, these data suggest that OPN could playa role in the neuroprotective/cone-reactivating effects of hRPCs. Theother factors tested are less likely to play such a role (unless theyhappen to be massively upregulated following transplantation in responseto the microenvironment of the vitreous/degenerative retina, which isless likely).

Fluorescence-activated cell sorting was carried out to characterizehRPCs and defining the extent of marker expression within thepopulation. Cultured cells or dissociated retinal single cells arestained by either conjugated surface antibody marker/isotype control(BD) or fixed and permeabilized, followed by conjugated intracellularantibody marker/isotype control (BD) staining for 30 min at roomtemperature. After washing in stain buffer (BD) 3 times, cells were runby BD Aria II Sorter. Data were obtained from same 3 simultaneous fetaleye donations used for the microarray studies.

FIG. 48 shows expression of 10 markers, either lineage related orpotentially relevant surface or genetic markers in retinal tissue at Day0 compared to cultured hRPCs grown under normoxic (20%) conditions orlow oxygen (3%) conditions. The data show differences between retinaltissue of origin and cultured RPCs, particularly massive upregulation ofMHC class I, accompanied by large increase in Fas (CD95), but not MHCclass II. GFAP is low but increased, to a lesser degree. Other markerschange in varying degrees.

Example 3 In vivo Efficacy of RPCs

RPCs were prepared for transplantation by first harvesting them withTrypLE Express and collecting them by centrifugation at 1000 rpm for 5minutes. Cells were washed once in HBSS, and then resuspended in coldHBSS to determine cell viability and cell number. For humantransplantation, 0.5×10⁶ cells in 100 μl HBSS were used. Fortransplantation into rats, varying doses ranging from 4000 to 75,000cells in 2 μl HBSS were used.

Human RPCs were transplanted as a suspension to the vitreous orsubretinal space of dystrophic hooded RCS rats. Rats were maintained oncyclosporin A and steroids to avoid rejection of xenografts. Controleyes received sham injections (subretinal, intravitreal) consisting ofvehicle alone. Grafted animals were tested functionally in theunrestrained waking state using a commercial apparatus designed forquantification of the optomotor response (OR). A subset of animals wastested for luminance threshold across the visual field. This was done byelectrophysiology via extracellular recordings in the contralateralsuperior colliculus. At the end of the study, eyes were collected,fixed, and analyzed histologically for evidence of host photoreceptorrescue and donor cell survival.

FIG. 49 illustrates proof of concept of methods of the invention usingin vivo transplantation: hRPCs (or vehicle alone, “sham”) were injectedto the eyes of dystrophic RCS rats (model of a hereditary photoreceptordegeneration). Injections were placed in either the vitreous orsubretinal space. Animals with hRPC grafts to either location performedsignificantly better than shams, or untreated controls, at age 60 days.Histology at that time point showed extensive rescue, which was locatedin the region of the subretinal injection (FIGS. 50-51), or widespreadacross the retina in the case of intravitreal grafts (FIGS. 52-53).Several cases were further examined for luminance threshold at age 90days, across the visual field, using electrophysiological recordings inthe contralateral superior colliculus. Animals with transplantsexhibited significantly improved sensitivity compared to shams oruntreated controls. Histology at 90 days showed persistent high levelrescue of host photoreceptors. FIGS. 54-55 show the results ofimmunocytochemistry performed on RCS whole mounts.

The in vivo data confirm and demonstrate that hRPC transplantation issuccessful at functional and anatomic levels in a rat model. The dataalso indicate that intravitreal injection can, in certain circumstances,have advantages over subretinal grafts in terms of the extent of hostretina that is rescued. RPCs are also effective when placedsubretinally, albeit in a more restricted manner (such restriction inrescue is the case with subretinal placement of most if not allnon-malignant cells used by variety of investigators).

Example 4 Clinical Study of hRPC Transplantation in Patients

A prospective, open-label, feasibility study of intraocular injectionsof RPCs in patients with retinal disease to obtain preliminary safetydata was carried out. Cells and tissues were first evaluated for safetyprior to commencement of the clinical study. Tissues were sourced fromAdvanced Bioscience Resources, Inc. (ABR), which is also able to provideGTP level tissue samples which are anticipated to form the basis ofestablishing a CGMP master cell bank based on this technology. Pathologyand blood testing of donor samples for exposure to adventitious agents(e.g., HIV, hepatitis B and C viruses, cytomegalovirus) was carried outand tests for endotoxin, mycoplasma, and fungi, such as the limulusamebocyte lysate (LAL) test (kinetic turbidimetric method) for endotoxindetection and the Fungitell kinetic chromogenic method for detection offungal contamination or final container sterility test by directinobulation, were performed on cultured donor samples. Cell culturemedium was collected and measured in our lab or mycoplasma core serviceusing LookOut Mycoplasma PCR Detection Kit (Sigma) or MycoAlertmycoplasma detection kit (Lonza). Cultured donor samples were alsosubjected to a soft agar assay to test for tumorigenic potential. Cellsuspensions of either 0.2×10⁵ or 1.0×10⁵ cells from different donationsand different time points were seeded into growth medium containing0.35% agar and then overlaid onto 0.7% agar gel. After incubation for 28days, colonies were stained with 0.005% crystal violet and scored forgrowth relative to positive and negative controls. Cells fortransplantation were also subjected to a test for telomerase activity.One microgram of cell protein from different donations and differenttime points in culture were tested using TRAPEZE RT Telomerase DetectionKit (Chemicon) with TITANIUM Taq DNA Polymerase (BD Clontech), accordingto manufacturer's instructions. In addition, cells were karyotyped by athird-party company to detect for the presence of any abnormalities, asthey can be indicative of spontaneous immortalization and cancerousbehavior of cultured progenitor cells. Finally, cell viability and cellnumber were determined by Trypan blue (Invitrogen) staining, counted byCountess automated cell counter (Invitrogen) or manually using ahemocytometer (Fisher Scientific).

Clinical lab results indicated that blood samples from donors werenegative for lethal viruses and other adventitious agents. Cells fortransplantation were negative for endotoxin, mycoplasma, and fungalinfection as well. In addition, cells prepared for transplant did notform colonies in soft agar, indicating their lack of tumorigenicity.Karyotype analysis was also performed to ensure clinical safety ofcultured hRPCs (was outsourced to established FDA-respected vendor, CellLine Genetics). Karyotyping revealed no abnormalities either. Withregard for cell viability and cell number, harvested cells were left intransplantation medium on ice out of incubator for various lengths oftime. A subset was also expelled through a 27 g hypodermic needle toevaluate the effect of that on survival. Expulsion through hypodermichad no detectible effect. The anticipated actual procedure time is lessthan 1 hour. Survival outside incubator began to drop appreciably at 2.5hr, but remained greater than 90% (acceptable) out to 3.5 hours. Thissuggested that the clinical transplantation procedure would not have amajor effect on donor cell survival.

Cells prepared for transplantation exhibit low or moderate, but notelevated levels of telomerase activity. Telomerase activity is typicallydown-regulated in mammalian cells beyond early embryonic development,except in certain cells including those of the germline which must beeffectively immortal and transmitted indefinitely. The normal loss oftelomerase activity is associated with both a limited lifespan for theorganism, but also indicates low probability of tumorigenicity.Increased telomerase activity is seen in mammalian cancer cells andpluripotent stem cells and immortalized human cells in culture. Thestrong association with malignant activity means that it is potentiallydangerous to transplant cells with elevated telomerase levels. Thesafety tests indicate that the conditions to prepare cells fortransplantation do not induce elevated telomerase activity, over a rangeof time points in culture out to 97 days (i.e., beyond current usablelimit). In fact, telomerase activity tends to drop off with time,consistent with developmental “senescence”. In other words, the cellseventually become senescent and lose the ability to proliferate.

Eligible patients received one unilateral injection of cells.Eligibility was determined by selecting for patients having any one ofsevere end stage retinal or optic nerve disease, poor residual centralvisual acuity of 20/200 or less with the use of a correcting lens,and/or dismal vision prognosis. Patients also must have adequate pupildilation and clear ocular media to permit stereoscopic fundusphotography, intraocular pressure of 21 mm Hg or less and open anteriorchamber angle. Patients were excluded if they exhibited narrow anteriorchamber angle, anterior chamber synechia or neovascularization, ahistory of angle closure glaucoma, significant existing media opacitiesthat would obstruct view during treatment, fundus examination,measurement of visual acuity, general evaluation of toxicity, anyintraocular surgery in the same eye within three months prior to studyentry; having known serious allergies to fluorescein dye used forangiography; a prior history or evidence of severe cardiac disease (NYHAFunctional Class III or IV), myocardial infarction within six months, orventricular tachyarrhythmias requiring continuing treatment, or unstableangina.

Three legally blind patients with retinal disease (i.e., retinitispigmentosa) and visual acuity no greater than 20/200 with a poorprognosis for improvement in vision were selected. Enrolled patientsranged in age from 46 to 57, two females, one male, all them with thediagnosis of RP. Two had IOL, one had a cataract.

Early passage fetal human retinal progenitor cells were characterized bytranscript and protein expression profiles and tested for normalkaryotype. The cells were also negative for fungi, bacteria (endotoxin),mycoplasma. Tissues were also screened for adventitious viruses and werefound to be negative for HIV1 and HIV2 antibodies, Hepatitis B antigens,Hepatitis C antibodies, syphilis, Herpes simplex virus IgM antibodies,West Nile Virus TMA:singlet, and EBV IgM VCA antibodies. Cells wereprepared for intravitral bolus injection at a dose of 100 μl cellsuspension (which delivered approximately 0.5 million cells to the eye).

For three days, the patients were self-treated with topical antibioticdrop at home prior to Day 0. On Day 0, patients were subjected to abaseline clinical examination, including fundus photography. Eyes wereirrigated and administered topical antibiotics, then pupils were dilatedand topical anesthesic drops applied. Awake, non-sedated patients wereinjected once with cells through the wall of the globe at the level ofthe “surgical limbus” into the vitreal cavity, using a standardangled-entry approach to create self-sealing entry passage and avoidreflux of graft under direct visualization by surgical microscope understerile operative conditions. An anterior chamber paracentesis wasperformed to prevent iatrogenic elevation of intraocular pressure. Noimmunosuppressive therapy, sutures, or wound closure procedure wereinvolved. Post-injection antibiotics were administered thereafter andintraocular pressure carefully monitored. All patients were dischargedsame day, with accompanying person and no hospital stay was required.Patients were advised to keep their heads elevated post-injection atleast 45 degrees for at least 2 hours, to avoid cellular bolus settlingover the macula. Prior to discharge from hospital, patients wereinstructed to sleep at home with their heads slightly elevated for 2-3days post-injection.

Clinical follow-ups were scheduled 1 day, 3 days, 1 week, 1 month, 2months, 3 months, 6 months, 1 year, and annually for 5 years aftertreatment. Incidence and severity of ocular adverse events wereidentified by standard ophthalmic examination techniques, includingfundus examination, best corrected visual acuity (BCVA), IOP, slit lampexamination, fluorescein angiography (FA), Optical Coherence Tomography(OCT), stereo-fundus photography, and cone flicker electroetinography(ERG) response.

Follow up slit lamp examination of all patients revealed that cells ofgraft can be visualized in the vitreous, some congealing intostrands-like structures, confirming that the transplanted cells hadentered eye and stayed there, and that vitreous placement resulted in acollection of vitreal cells. Notably, this did not negate visualimprovement due to obscuration of visual axis (blocking subjects viewout of the eye) as might theoretically occur.

A follow up B Scan was carried out, wherein an ultrasound devicefollowed vitreal cells at 4 months post-operation. Results show no tumorformation in the eye and no tumor formation in the anterior orbit(behind the eye). The persistence of grafted cells (and persistence ofvisual improvements) suggests that there is no need for routine immunesuppression in patients receiving the transplanted cells. No evidence oftumors, vascular complications or retinal detachment was seen either onexamination or by fundus photography. Notably, no patient experiencedloss of vision due to procedure and all patients reported detectableimprovement in vision. Improvements were related to severity of disease:the worse the initial vision, the more limited the improvement, whilethe better the initial vision, the greater and more rapid theimprovement. One patient (002) with hand motion vision was able to seethe eye chart again at 4 months. Another patient (003) regained centralfixation and a degree of macular function (which is needed to achievevisual acuity better than 20/200). Patient 003 achieved an improvementin visual acuity of “20 letters” on the eye chart, which is a very largeand significant improvement. Visual improvements were sustained andacuity gains persisted to at least 1.5 years. Patients also reportedbetter visual function in general and improved activities of dailyliving.

The results of the visual acuity test as used for comparison of trendbetween patients is provided in FIG. 55. Neither scale is preciselylinear. Evidence of rapid improvement in vision was observed within the1s^(t) week in all three cases, most consistent with a clinicallysignificant trophic effect on host retina. Also, tendency towardadditional improvements were seen after 1 month, which may includeengraftment and retina cell replacement. Some issues with the patientscorrespond to certain obvious deflections in the data trends. Patient002 (blue) was non-compliant with post-operative drops and developed ananterior uveitis that rapidly resolved with standard post-opmedications. Notably, her long-term progress shows improvement. Patient001 (pink) had a pre-existing cataract which worsened over the first fewmonths post-transplantation, perhaps due to the procedure, and that mayhave related to her secondary decline of vision (although vision wasimproved over the initial level). No abnormalities of IOP were found tobe associated with transplantation of hRPCs (See Table 1).

TABLE 1 Intraocular Pressures of Study Eyes Intraocular Pressure (mm Hg)Patient No. Initial Final 001 Normal Normal 002 Normal Normal 003 NormalNormal

In a visual field test, all 3 patients had lost central fixation priorto treatment because of poor visual acuity associated with loss ofmacular function due to end stage retinitis pigmentosa. Patient 003regained central fixation at day 3, and was able to perform automatedvisual field testing which revealed a small 2-degree area with 20DBsensitivity (normal). Visual sensitivity in this patient was alsoelevated in another area nasal to the foveal fixation point. Since thispatient regained central fixation, it was possible to test his visualfield using automated perimetry (HVF). This test showed that he hadregained a small, but highly sensitive island of vision in the centralfoveal region. Data supports the concept that this treatment involves arapid trophic effect from the grafted cells, distributed to the retinathrough the vitreous body, and which appears to restore function toresidual host cones. Reports of improved night vision (multiplepatients) and improved ERG performance (patient 003) support a role forimproved rod photoreceptor function as well. These improvements may alsobe due to functional cellular integration into the host retina. Otherretinal exams such as retina topography, RNFL, OCT, and others wereperformed. No evidence of tumor formation or immunological tissuerejection were seen.

In summary, all patients in the clinical trial experienced improvedvisual acuity with treatment. Visual benefits persisted for at least 20months post-injection. At least one patient had improved visual fieldcharacterized by a return of central fixation. No surgical complicationsor evidence of immune rejection were observed, despite the lack ofimmune suppression or administration of immunosuppressant drugs. Nosignificant donor cell proliferation was seen in vivo, based on allclinical examinations, i.e., slitlamp, indirect ophthalmoscopy,ultrasound (B scan) of eye and orbit, and fundus photographs. No tumorformation was seen for up to at least 20 months.

Although a few variations have been described in detail above, othermodifications or additions are possible. In particular, further featuresand/or variations may be provided in addition to those set forth herein.For example, the implementations described above may be directed tovarious combinations and subcombinations of the disclosed featuresand/or combinations and subcombinations of several further featuresdisclosed above. In addition, the logic flow depicted in theaccompanying figures and/or described herein does not require theparticular order shown, or sequential order, to achieve desirableresults. Other embodiments may be within the scope of the followingclaims.

1. An isolated cell population produced by: harvesting human retinaltissue at a stage after which the retina is formed but beforephotoreceptor outer segments are fully formed throughout the retina andbefore retinal vascularization substantially completed or completed,wherein the tissues are harvested from a human retina at a gestationalage between about 12 weeks to about 28 weeks; dissociating the harvestedtissues mechanically and enzymatically to generate a dissociatedsuspension of cells and cell clusters; and culturing the dissociatedsuspension of cells at atmospheric oxygen level or at low oxygenconditions that mimic oxygen levels of a developing fetal retina duringgestation, or under hypoxic conditions, in serum free culture medium inculture flasks or plates coated with a xeno-free fibronectin, anornithine, a polylysine, a laminin, or an equivalent thereof, for nomore than 10 passages, wherein vitamin C is added to the culture mediaevery 1 or 2 days in an amount between 0.01 mg/ml to 0.5 mg/ml: whereinafter the culturing the human retinal progenitor cells express one ormore markers selected from the group consisting of nestin, Sox2, Ki-67,MHC Class I, and Fas/CD95, wherein nestin is expressed by greater than90% of the cells in the population, wherein Sox2 is expressed by greaterthan 80% of the cells in the population, wherein Ki-67 is expressed bygreater than 30% of the cells in the population, wherein MHC Class I isexpressed by greater than 70% of the cells in the population, andwherein Fas/CD95 is expressed by greater than 30% of the cells in thepopulation, thereby making an isolated cell population wherein cells inthe population express one or more markers selected from the groupconsisting of nestin, Sox2, Ki-67, MHC Class I, and Fas/CD95.
 2. Theisolated cell population of claim 1, wherein the cells in the populationfurther express one or more markers selected from the group consistingof vimentin, CD9, CD81, AQP4, CXCR4, CD15/LeX/SSEA1, GD2 ganglioside,CD133, β3-tubulin, MAP2, GFAP, OPN/SPP1, PTN, KDR, and TEK.
 3. Theisolated cell population of claim 1, wherein the enzymatic dissociatingcomprises trypsin.
 4. The isolated cell population of claim 1, whereinthe xeno-free fibronectin comprises human plasma fibronectin.
 5. Theisolated cell population of claim 1, wherein the isolated cellpopulation is cryopreserved.
 6. The isolated cell population of claim 1,wherein the cells in the isolated cell population express on theirsurface a Sox2, a Ki-67, an MHC Class I, and a Fas/CD95.
 7. The isolatedpopulation of claim 1, wherein the low oxygen conditions that mimicoxygen levels of a developing fetal retina during gestation is about 3%oxygen.
 8. A method of making a formulation, a product of manufacture,or a composition comprising a heterogeneous mixture of non-immortalhuman fetal neural retinal cells, wherein the method comprises: (a)harvesting human retinal tissue at a stage after which the retina isformed but before photoreceptor outer segments are fully formedthroughout the retina and before retinal vascularization substantiallycompleted or completed, wherein the tissues are harvested from a humanretina at a gestational age between about 12 weeks to about 28 weeks;(b) dissociating the harvested tissues mechanically and enzymatically togenerate a dissociated suspension of cells and cell clusters; and (c)culturing the dissociated suspension of cells and cell clusters in serumfree culture medium in culture flasks or plates coated with a xeno-freefibronectin, an ornithine, a polylysine, a laminin, or an equivalentthereof, for no more than 10 passages, wherein the cells are passaged atbetween about 40% to 90% confluence and treated with an enzyme at eachpassage to dissociate the cells, wherein the culture media is changedabout every 1 to 2 days, and wherein vitamin C is added to the culturemedia every 1 or 2 days in an amount between 0.01 mg/ml to 0.5 mg/ml. 9.The method of claim 8, wherein the enzymatic dissociating comprisestrypsin.
 10. The method of claim 8, wherein the xeno-free fibronectincomprises human plasma fibronectin.
 11. The method of claim 8, whereinthe cells are cryopreserved.
 12. The method of claim 8, wherein thecells and cell cultures are cultured in a culture media.
 13. The methodof claim 12, wherein the culture media comprises supplements oradditives that support cell survival or growth.
 14. The method of claim13, wherein the supplements or additives that support cell survival orgrowth are selected from the group consisting of L-glutamine, humanrecombinant growth factors consisting of EGF and bFGF, and other growthfactors.
 15. The method of claim 8, wherein the cells and cell clustersare cultured or grown under low oxygen conditions, or oxygen conditionsthat approximate or closely mimic oxygen levels of a developing fetalretina during gestation, or at about 2%, 2.5%, 3%, 3.5% oxygen.
 16. Themethod of claim 8, wherein the media is supplemented with albumin, orrecombinant albumin in an amount to have an initial concentration ofabout 1.0 mg/ml.
 17. The method of claim 8, wherein the sample of cellsor cell clusters is screened for the presence of a pathogen, a bacteria,an endotoxin, a fungus, a mycoplasma, a virus, a hepatitis virus or anHIV virus.
 18. The method of claim 8, wherein the sample of cells orcell clusters is screened for the presence of a normal karyotype. 19.The method of claim 8, wherein the sample of cells or cell clusters doesnot exhibit elevated telomerase activity.
 20. The method of claim 8,wherein the sample of cells or cell clusters is screened for viability.21. The method of claim 8, wherein the sample of cells or cell clustersis screened for tumorigenicity.
 22. The method according to claim 8,wherein the method for making the heterogeneous mixture of non-immortalhuman fetal neural retinal cells further comprises: selecting humanfetal neural retinal cells on the basis of cell surface or geneticmarkers, or selecting human fetal neural retinal cells on the basis of ahuman fetal neural retinal cell transcriptome profile, proteome profileand/or a genomic profile.
 23. The method of claim 22, wherein the stepof selecting human fetal neural retinal cells on the basis of cellsurface or genetic markers further comprises selecting the cells eitherbefore culturing, or selecting the cells prospectively, or selecting thecells after culturing, or selecting the cells both before culturing andafter culturing.
 24. A method for treating a retinal degeneration in asubject in need thereof comprising administering to the subject aneffective amount of a composition comprising non-immortal human fetalretinal progenitor cells, wherein the effective amount of thecomposition comprises between 1000 and 10 million cells per dose and thecomposition is injected into a vitreous cavity of the subject, whereinthe composition is prepared by: (a) harvesting a sample of cellscomprising a plurality of human fetal neural retinal cells from human atabout 12 weeks to about 28 weeks gestational age; (b) mechanically,enzymatically, or mechanically and enzymatically digesting the harvestedsample of cells to make a dissociated suspension of cells and cellclusters; and (c) culturing the suspension in serum-free media inculture flasks or plates coated with a xeno-free fibronectin, andornithine, a polylysine, or a laminin, for no more than 10 passages,wherein the cells are passaged at between about 40 to 90% confluence andtreated with an enzyme at each passage to dissociate the cells andbetween 0.01 mg/ml and 0.5 mg/ml vitamin C is added to the culture mediaevery 1 to 2 days, thereby making nonimmortal human retinal progenitorcells and increasing human retinal progenitor cell yield by about 30%,wherein the non-immortal human retinal progenitor cells express one ormore markers selected from the group consisting of nestin, Sox2, Ki-67,MHC Class I, and Fas/C D95, wherein nestin is expressed by greater than90% of the cells in the population, wherein Sox2 is expressed by greaterthan 80% of the cells in the population, wherein Ki-67 is expressed bygreater than 30% of the cells in the population, wherein MHC Class I isexpressed by greater than 70% of the cells in the population, andwherein Fas/CD95 is expressed by greater than 30% of the cells in thepopulation, wherein the injected non-immortal human retinal progenitorcells provide neuroprotective trophic or regenerative influences withoutintegration into the subject, thereby treating the retinal degeneration.25. The method of claim 24, wherein the subject is a human or anon-human mammal.
 26. The method of claim 24, wherein the retinaldegeneration comprises or is the result of retinitis pigmentosa (RP).27. The method of claim 24, wherein the cells in the composition furtherexpress one or more markers selected from the group consisting ofvimentin, CD9, CD81, AQP4, CXCR4, CD15/LeX/SSEA1, GD2 ganglioside,CD133, β3-tubulin, MAP2, GFAP, OPN/SPP1, PTN, KDR, and TEK.
 28. Themethod of claim 24, further comprising measuring changes in vision inthe subject after administering to the subject the effective amount ofthe composition.
 29. The method of claim 24, wherein the retinaldegeneration comprises or is the result of Usher's syndrome.
 30. Themethod of claim 24, wherein the retinal degeneration comprises arod-cone or cone-rod dystrophy.
 31. The method of claim 24, wherein theretinal degeneration comprises or is the result of a ciliopathy.
 32. Themethod of claim 24, wherein the retinal degeneration comprises or is theresult of age related macular degeneration (AMD), wet AMD, or dry AMD.33. The method of claim 24, wherein the retinal degeneration comprisesor is the result of a retinal photoreceptor disease.